Polyspecific immunoconjugates and antibody compositiesfor targeting the multidrug resistant phenotype

ABSTRACT

Polyspecific immunoconjugates and antibody composites that bind a multidrug transporter protein and an antigen associated with a tumor or infectious agent are used to overcome the multidrug resistant phenotype. These immunoconjugates and composites also can be used diagnostically to determine whether the failure of traditional chemotherapy is due to the presence of multidrug resistant tumor cells, multidrug resistant HIV-infected cells or multidrug resistant infectious agents.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to novel polyspecificimmunoconjugates that are useful for diagnosis and therapy of diseasescaused by cells that are multidrug resistant. In particular, thisinvention relates to polyspecific immunoconjugates that comprise atleast one moiety that binds with a multidrug transporter protein, atleast one moiety that binds with a tumor associated antigen orinfectious agent antigen, and a therapeutic or diagnostic agent. Thisinvention also relates to methods of diagnosis and therapy using thepolyspecific immunoconjugates. This invention further relates todiagnostic and therapeutic uses of antibody composites comprising atleast one moiety that binds with a multidrug transporter protein, and atleast one moiety that binds with a tumor associated antigen orinfectious agent antigen.

[0003] 2. Background

[0004] One of the major limitations of cancer chemotherapy is thedevelopment of drug resistance by cancer cells. Despite initialsensitivity to a particular chemotherapeutic agent, some tumors becomeprogressively unresponsive to the particular agent, or to variouschemotherapeutic agents. This phenomenon of acquired drug resistance isbelieved to be due to the selection and growth of drug resistant mutanttumor cells. See, for example, Deuchars et al., Sem. Oncol. 16: 156(1989).

[0005] Cultured cell lines and transplantable tumors have been used tostudy the mechanism of acquired drug resistance in vitro. These studieshave shown that under certain selection conditions, cells may acquiresimultaneous resistance to a diverse group of drugs that are unrelatedto the selecting agent in structure, cellular target and mode of action.See, for example, Bradley et al., Biochim. Biophys. Acta 948: 87 (1988);Deuchars et al., supra. Many of the drugs affected by this“multidrug-resistance” (MDR) phenotype are important in currenttreatment protocols, such as vincristine, actinomycin D, and adriamycin.Id.

[0006] The MDR phenotype is consistently associated with over-expressionof a 170 kilodalton membrane glycoprotein, designated “gp170” or“P-glycoprotein.” Endicott et al., Ann. Rev. Biochem. 58: 137 (1989);Kane et al., J. Bioenerg. Biomembr. 22: 593 (1990); Efferth et al.,Urol. Res. 18: 309 (1990). Studies indicate that P-glycoprotein is atransmembrane protein responsible for an ATP-dependent efflux of a broadspectrum of structurally and functionally distinct drugs frommultidrug-resistant cells. Riordan et al., Pharmacol. Ther. 28: 51(1985). In fact, expression of P-glycoprotein has been shown to bepredictive of a poor response to chemotherapy in a number of neoplasms.See, for example, Pearson et al., J. Nat'l Cancer Inst. 83: 1386 (1991).

[0007] Recent observations indicate that infectious agents can inducethe MDR phenotype in noncancerous cells. For example, prolongedtreatment with 3′-azido-3′-deoxythymidine (AZT) for humanimmunodeficiency virus (HIV) infection is associated with an acquiredresistance to AZT. Gollapudi et al., Biochem. Biophys. Res. Commun. 171:1002 (1990); Antonelli et al., AIDS Research and Human Retroviruses 8:1839 (1992). In vitro studies demonstrate that HIV-infected human cellshave an increased expression of P-glycoprotein and accumulate less AZT,compared with non-infected control cells. Id.; Gupta et al., J. Clin.Immunol. 13: 289 (1993). Thus, overexpression of P-glycoprotein and theaccompanying MDR phenotype can impair chemotherapy with anti-viraldrugs.

[0008] Considerable effort has been employed to overcome themultidrug-resistant phenotype and thus, improve the efficacy ofchemotherapy. Most of these strategies have involved pharmacologicalagents that enhance the intracellular accumulation of the cancer drugsby biochemically inhibiting the multidrug transporter. See, for example,Ford et al., Pharmacol. Rev. 42: 155 (1990). Examples of agents thatmodulate P-glycoprotein activity include calcium channel blockers,calmodulin inhibitors, antiarrythmics, antimalarials, variouslysoosmotropic agents, steroids, antiestrogens, and cyclic peptideantibiotics. Rittmann-Grauer et al., Cancer Res. 52: 1810 (1992).

[0009] However, multidrug-resistant reversing drugs used in earlyclinical trials have shown major side effects unrelated to theinhibition of P-glycoprotein, such as cardiac toxicity (verapamil) orimmunosuppression (cyclosporin A), which limit the dosage of drug thatcan be administered. See, for example, Ozols et al., J. Clin. Oncol. 5:641 (1987); Dalton et al., J. Clin. Oncol. 7: 415 (1989); Cano-Gauci etal., Biochem. Pharmacol. 36: 2115 (1987); Ford et al., supra. Thus,there has been limited success in reversing MDR in vivo due to thetoxicity of many of these small modulators. See, for example,Rittmann-Grauer et al., supra.

[0010] The use of antibody-drug conjugates provides an alternativeapproach to overcoming the MDR phenotype. For example, in vitro studieshave shown that MDR can be partially overcome by conjugating theresistant drug to an antitumor antibody to increase uptake andsubsequent cell death. Durrant et al., Brit. J. Cancer 56: 722 (1987);Sheldon et al., Anticancer Res. 9: 637 (1989). This approach, however,lacks specificity for tumor cells that express the MDR phenotype.

[0011] A more targeted approach to overcoming the MDR phenotype is touse antibodies or antibody conjugates that bind with P-glycoprotein. Forexample, the administration of an anti-P-glycoprotein monoclonalantibody and a resistant drug can increase the survival time of nudemice that carry human tumor cells. Pearson et al., J. Nat'l Cancer Inst.83: 1386 (1991); Iwahashi et al., Cancer Res. 53: 5475 (1993). Also, seeGrauer et al., international publication No. WO 93/02105 (1993). Inaddition, an anti-P-glycoprotein monoclonal antibody-Pseudomonas toxinconjugate has been shown to kill multidrug-resistant human cells invitro. FitzGerald et al., Proc. Nat'l Acad. Sci. USA 84: 4288 (1987).Also, see Efferth et al., Med. Oncol. & Tumor Pharmacother. 9: 11(1992), and Mechetner et al., international publication No. WO 93/19094(1993).

[0012] Similarly, investigators have produced bispecific antibodiescomprising a P-glycoprotein binding moiety and a moiety that binds witha cytotoxic cell. van Dijk et al., Int. J. Cancer 44: 738 (1989); Ringet al., international Publication No. WO 92/08802 (1992). The theorybehind this approach is that the bispecific antibodies can be used todirect cytotoxic cells to multidrug-resistant cells that expressP-glycoprotein.

[0013] However, studies have shown that P-glycoprotein is expressed innormal human tissues, such as liver, kidney, adrenal gland, pancreas,colon and jejunum. See, for example, Endicott et al., Ann. Rev. Biochem.58: 137 (1989). Consequently, investigators have warned that “blockingP-glycoprotein action in order to circumvent MDR will also affect thenormally expressed P-glycoprotein and this may cause unacceptable sidetoxic effects.” Childs et al., “The MDR Superfamily of Genes and ItsBiological Implications,” in IMPORTANT ADVANCES IN ONCOLOGY 1994, DeVitaet al., (eds.), pages 21-36 (J.B. Lippincott Co. 1994). This admonitionparticularly applies to therapeutic methods that use antibody conjugatesconsisting of a P-glycoprotein binding moiety and a cytotoxic agent.Therefore, the success of an antibody-directed treatment of MDR tumorswill mainly depend upon the ability to kill drug-resistant tumor cellswith tolerable side effects to normal tissues of the patient. Efferth etal., Med. Oncol. & Tumor Pharmacother. 9: 11 (1992).

[0014] Thus, an need exists for a method to overcome the MDR phenotypebut that also minimizes toxicity to normal tissue.

[0015] The emergence of the MDR phenotype also is the major cause offailure in the treatment of infectious diseases. Davies, Science 264:375 (1994). In particular, pathogenic bacteria have active drug effluxsystems of very broad substrate specificity. Nikaido, Science 264: 382(1994), which is incorporated by reference. For example, studiesindicate that a drug efflux system plays a major role in the intrinsicresistance of Psuedomonas aeruginosa, a common opportunistic pathogen.Poole et al., Mol. Microbiol. 10: 529 (1993); Poole et al., J.Bacteriol. 175: 7363 (1993).

[0016] Recent studies indicate that bacterial drug efflux systems arefunctionally similar to the mammalian MDR efflux pump. As anillustration, both the Bacillus subtilis and the mammalian multidrugtransporters can be inhibited by reserpine and verapamil. Neyfakh etal., Proc. Nat'l Acad. Sci. 88: 4781 (1991). Moreover, investigatorshave recognized a superfamily of ATP-dependent membrane transportersthat includes prokaryotic permeases and mammalian P-glycoprotein. Doigeet al., Ann. Rev. Microbiol. 47: 291 (1993).

[0017] Active drug efflux as a mechanism for drug resistance issignificant in nonbacterial infectious agents. For instance, aPlasmodium falciparum protein is involved in imparting resistance toquinoline-containing drugs used for prophylaxis and treatment ofmalaria. Id.; Bray, FEMS Microbiol. Lett. 113: 1 (1993). In addition,drug resistance has been linked to active efflux in the fungus,Aspergillus nidulans. de Waard et al., Pestic. Biochem. Physiol. 13: 255(1980).

[0018] Historically, the pharmaceutical industry has concentrated ondesigning drugs to overcome specific mechanisms of MDR in infectiousagents, such as increased degradation of particular drugs andinactivation of drugs by enzymatic modification of specific groups.Nikaido et al., supra. However, in the future, general mechanisms ofMDR, such as active drug efflux, are likely to become more important inthe clinical setting.

[0019] Thus, a need exists for methods that can be used to inhibit thefunction of multidrug transporter proteins expressed by infectiousagents.

SUMMARY OF THE INVENTION

[0020] Accordingly, it is an object of the present invention to providea method for overcoming the multidrug-resistant phenotype that has atherapeutic index superior to conventional methods.

[0021] Another object of this invention is to provide methods forselectively targeting diagnostic and therapeutic agents tomultidrug-resistant cells, while avoiding major toxic side effects tonormal organs.

[0022] Another object of this invention is to provide antibodycomposites that bind a multidrug transporter protein and an antigenassociated with a tumor or infectious agent.

[0023] A further object of this invention is to provide polyspecificimmunoconjugates which are conjugates of antibody composites anddiagnostic or therapeutic agents.

[0024] These and other objects are achieved, in accordance with oneembodiment of the present invention by the provision of a polyspecificimmunoconjugate comprising:

[0025] (a) at least one antibody component that binds with a firstepitope of a multidrug transporter protein;

[0026] (b) at least one antibody component that binds with a firstepitope of an antigen, wherein the antigen is associated with a tumor oran infectious agent; and

[0027] (c) at least one diagnostic or therapeutic agent.

[0028] The antibody components of such a polyspecific immunoconjugateare selected from the group consisting of (a) a murine monoclonalantibody; (b) a humanized antibody derived from (a); (c) a humanmonoclonal antibody; (d) a subhuman primate antibody; and (e) anantibody fragment derived from (a), (b), (c) or (d), wherein theantibody fragment is selected from the group consisting of F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, sFv and minimal recognition unit. The multidrugtransporter protein of such a polyspecific immunoconjugate is selectedfrom the group consisting of P-glycoprotein, OtrB, Tel(L), Mmr, ActII,TcmA, NorA, QacA, CMlA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE,MvrC, MsrA, DrrA, DrrB, TlrC, Bmr, TetA and OprK.

[0029] As stated above, the polyspecific immunoconjugate comprises adiagnostic or therapeutic agent. A suitable diagnostic agent is selectedfrom the group consisting of radioactive label, photoactive agent ordye, florescent label, enzyme label, bioluminescent label,chemiluminescent label, colloidal gold and paramagnetic ion. Moreover, asuitable radioactive label may be a γ-emitter or a positron-emitter.Preferably, γ-emitters have a gamma radiation emission peak in the rangeof 50-500 Kev, such as a radioisotope selected from the group consistingof ^(99m)Tc, ⁶⁷Ga, ¹²³I, ¹²⁵I and ¹³¹I.

[0030] A suitable therapeutic agent is selected from the groupconsisting of radioisotope, boron addend, immunomodulator, toxin,photoactive agent or dye, cancer chemotherapeutic drug, antiviral drug,antifungal drug, antibacterial drug, antiprotozoal drug andchemosensitizing agent. Moreover a suitable therapeutic radioisotope isselected from the group consisting of α-emitters, β-emitters,γ-emitters, Auger electron emitters, neutron capturing agents that emitα-particles and radioisotopes that decay by electron capture.Preferably, the radioisotope is selected from the group consisting of¹⁹⁸Au, ³²P, ¹²⁵I, ¹³¹I, ^(9O)Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu and ²¹¹At.

[0031] The present invention also contemplates polyspecificimmunoconjugates which further comprise an antibody component that bindswith a second epitope of the multidrug transporter protein. Moreover,polyspecific immunoconjugates may additionally comprise an antibodycomponent that binds with a second epitope of the tumor or infectiousagent associated antigen, or with an epitope of a second antigenassociated with the tumor or the infectious agent.

[0032] The present invention also is directed to a method for treating amammal having either a multidrug resistant tumor that expresses a tumorassociated antigen or a multidrug resistant disease caused by aninfectious agent, the method comprising the step of administering apolyspecific immunoconjugate to the mammal, wherein the polyspecificimmunoconjugate comprises:

[0033] (a) at least one antibody component that binds with a firstepitope of a multidrug transporter protein,

[0034] (b) at least one antibody component that binds with a firstepitope of an antigen, wherein the antigen is associated with the tumoror the infectious agent, and

[0035] (c) at least one therapeutic agent.

[0036] Moreover, the present invention contemplates methods furthercomprising the administration of a chemosensitizing agent orimmunomodulator to the mammal.

[0037] In addition, the present invention is directed to a method fordetecting the location of multidrug resistant (MDR) tumor cells, MDRHIV-infected cells or MDR infectious agents in a mammal having amultidrug resistant disease caused by a tumor or infectious agent, themethod comprising the steps of:

[0038] (a) parenterally injecting the mammal with an antibody compositecomprising (1) at least one antibody component that binds a firstepitope of a multidrug transporter protein, and (2) at least oneantibody component that binds a first epitope of an antigen that isassociated with the tumor or the infectious agent, wherein the antibodycomposite is conjugated with a biotin-binding molecule or with biotin;

[0039] (b) parenterally injecting a clearing composition comprised of:

[0040] (i) biotin, when the antibody composite is conjugated with abiotin-binding molecule, or

[0041] (ii) a biotin-binding molecule, when the antibody composite isconjugated with biotin,

[0042] and allowing the clearing composition to substantially clear theantibody composite from sites that do not contain MDR tumor cells, MDRHIV-infected cells or MDR infectious agents;

[0043] and

[0044] (c) parenterally injecting a diagnostic composition comprised of:

[0045] (i) biotin, when the antibody composite is conjugated with abiotin-binding molecule, or

[0046] (ii) a biotin-binding molecule, when the antibody composite isconjugated with

[0047] and a diagnostic agent which is conjugated with the biotin or thebiotin-binding molecule.

[0048] In such a detection method, the diagnostic agent is selected fromthe group consisting of radioactive label, photoactive agent or dye,fluorescent label and paramagnetic ion. Moreover, the biotin-bindingmolecule is avidin or streptavidin.

[0049] The present invention also contemplates a method for treating amammal having a multidrug resistant disease caused by a tumor orinfectious agent, the method comprising the steps of:

[0050] (a) parenterally injecting the mammal with an antibody compositecomprising (1) at least one antibody component that binds a firstepitope of a multidrug transporter protein, and (2) at least oneantibody component that binds a first epitope of an antigen that isassociated with the tumor or the infectious agent, wherein the antibodycomposite is conjugated with a biotin-binding molecule or with biotin;

[0051] (b) parenterally injecting a clearing composition comprised of:

[0052] (i) biotin, when the antibody composite is conjugated with abiotin-binding molecule, or

[0053] (ii) a biotin-binding molecule, when the antibody composite isconjugated with biotin,

[0054] and allowing the clearing composition to substantially clear theantibody composite from sites that do not contain multidrug resistant(MDR) cells or MDR infectious agents; and

[0055] (c) parenterally injecting a therapeutic composition comprisedof:

[0056] (i) biotin, when the antibody composite is conjugated with abiotin-binding molecule, or

[0057] (ii) a biotin-binding molecule, when the antibody composite isconjugated with biotin,

[0058] and a therapeutic agent which is conjugated with the biotin orthe biotin-binding molecule.

[0059] A suitable therapeutic agent is selected from the groupconsisting of radioisotope, boron addend, toxin, immunomodulator,photoactive agent or dye, cancer chemotherapeutic drug, antiviral drug,antifungal drug, antibacterial drug, antiprotozoal drug and achemosensitizing agent. Again, the biotin-binding molecule is avidin orstreptavidin.

[0060] The present invention also is directed to a method for detectingthe presence of multidrug resistant (MDR) tumor cells, MDR HIV-infectedcells or MDR infectious agents in a mammal, the method comprising:

[0061] (a) removing from the mammal a biological sample that issuspected of containing MDR tumor cells, MDR HIV-infected cells or MDRinfectious agents;

[0062] (b) contacting the biological sample with an antibody compositewhich comprises (1) at least one antibody component that binds with afirst epitope of a multidrug transporter protein, and (2) at least oneantibody component that binds with a first epitope of an antigen that isassociated with the tumor or the infectious agent, wherein thecontacting is performed under conditions which allow the binding of theantibody composite to the biological sample; and

[0063] (c) detecting any of the bound antibody composite.

[0064] Here, a suitable diagnostic agent selected from the groupconsisting of radioisotope, fluorescent label, chemiluminescent label,enzyme label, bioluminescent label and colloidal gold. Moreover, theantibody composite can further comprise biotin or a biotin-bindingmolecule.

[0065] The present invention is further directed to a method fordetecting the location of multidrug resistant (MDR) tumor cells, MDRHIV-infected cells or MDR infectious agents in a mammal having amultidrug resistant disease caused by a tumor or infectious agent, themethod comprising the steps of:

[0066] (a) parenterally injecting the mammal with a polyspecificimmunoconjugate that comprises (1) at least one antibody component thatbinds with a first epitope of a multidrug transporter protein, (2) atleast one antibody component that binds with a first epitope of anantigen that is associated with the tumor or infectious agent, and (3) adiagnostic agent;

[0067] (b) parenterally injecting the mammal with an antibody orantibody fragment that binds with the polyspecific immunoconjugate in anamount that is sufficient to decrease the level of circulatingpolyspecific immunoconjugate by about 10-85% within 2 to 72 hours;

[0068] (c) scanning the mammal with a detector to locate the site orsites of uptake of the polyspecific immunoconjugate.

[0069] A suitable diagnostic agent is selected from the group consistingof radioactive label, photoactive agent or dye, fluorescent label andparamagnetic ion.

[0070] The present invention also contemplates a method for treating amammal having a multidrug resistant disease caused by a tumor orinfectious agent, the method comprising the steps of:

[0071] (a) parenterally injecting the mammal with a polyspecificimmunoconjugate comprising (1) at least one antibody component thatbinds with a first epitope of a multidrug transporter protein, (2) atleast one antibody component that binds with a first epitope of anantigen that is associated with the tumor or infectious agent, and (3) atherapeutic agent; and

[0072] (b) parenterally injecting the mammal with an antibody orantibody fragment that binds with the polyspecific immunoconjugate in anamount that is sufficient to decrease the level of circulatingpolyspecific immunoconjugate by about 10-85% within 2 to 72 hours.

[0073] In addition, the present invention is directed to a method fordetecting the location of multidrug resistant (MDR) tumor cells, MDRHIV-infected cells or MDR infectious agents in a subject having amultidrug resistant disease caused by a tumor or infectious agent, themethod comprising the steps of:

[0074] (a) parenterally injecting the subject with a polyspecificimmunoconjugate comprising (1) at least one antibody component thatbinds with a first epitope of a multidrug transporter protein, (2) atleast one antibody component that binds with a first epitope of anantigen that is associated with a tumor or infectious agent, and (3) adiagnostic agent;

[0075] (b) surgically exposing or endoscopically accessing the interiorof the body cavity of the subject; and

[0076] (c) scanning the interior body cavity with a detection probe todetect the sites of accretion of the polyspecific immunoconjugate.

[0077] Suitable diagnostic agents include radioisotopes, such as aγ-emitter or a positron-emitter, and a photoactive agent or dye that isdetected by laser-induced fluorescence.

[0078] The present invention also contemplates a method for treating asubject having a multidrug resistant disease caused by a tumor orinfectious agent, the method comprising the steps of:

[0079] (a) parenterally injecting the subject with a polyspecificimmunoconjugate comprising (1) at least one antibody component thatbinds with a first epitope of a multidrug transporter protein, (2) atleast one antibody component that binds with a first epitope of anantigen that is associated with a tumor or infectious agent, and (3) aphotoactive agent or dye;

[0080] (b) surgically exposing or endoscopically accessing the interiorof the body cavity of the subject; and

[0081] (c) treating sites of accretion of the polyspecificimmunoconjugate to light, wherein the treatment activates thephotoactive agent or dye.

[0082] In addition, the present invention is directed to an antibodycomposite comprising:

[0083] (a) at least one antibody component that binds with a firstepitope of a multidrug transporter protein; and

[0084] (b) at least one antibody component that binds with a firstepitope of an antigen, wherein the antigen is associated with a tumor oran infectious agent.

[0085] Suitable antibody components of antibody composites are selectedfrom the group consisting of (a) a murine monoclonal antibody; (b) ahumanized antibody derived from (a); (c) a human monoclonal antibody;(d) a subhuman primate antibody; and (e) an antibody fragment derivedfrom (a), (b), (c) or (d), where an antibody fragment is selected fromthe group consisting of F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv and minimalrecognition unit. Moreover, a suitable multidrug transporter protein isselected from the group consisting of P-glycoprotein, OtrB, Tel(L), Mmr,ActII, TcmA, NorA, QacA, CmlA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr,QacE, MvrC, MsrA, DrrA, DrrB, TlrC, Bmr, TetA and OprK.

[0086] The present invention also contemplates an antibody compositefurther comprising an antibody component that binds with a secondepitope of the multidrug transporter protein. An antibody composite canadditionally include an antibody component that binds with a secondepitope of the tumor or infectious agent associated antigen, or with anepitope of a second antigen associated with the tumor or the infectiousagent.

[0087] The present invention is further directed to a method fortreating a mammal having either a multidrug resistant tumor thatexpresses a tumor associated antigen or a multidrug resistant diseasecaused by an infectious agent, the method comprising the step ofadministering an antibody composite to the mammal, wherein the antibodycomposite comprises:

[0088] (a) at least one antibody component that binds with a firstepitope of a multidrug transporter protein, and

[0089] (b) at least one antibody component that binds with a firstepitope of an antigen, wherein the antigen is associated with the tumoror the infectious agent.

[0090] Moreover, the present invention contemplates a method furthercomprising the step of administering a therapeutic agent to the mammal,wherein the therapeutic agent is selected from the group consisting ofcancer chemotherapeutic drug, antiviral drug, antifungal drug,antibacterial drug and antiprotozoal drug. Finally, the presentinvention also is directed to a method which further comprises the stepof administering an immunomodulator, wherein the immunomodulator isselected from the group consisting of cytokine, stem cell growth factorand hematopoietic factor.

DETAILED DESCRIPTION

[0091] 1. Definitions

[0092] In the description that follows, a number of terms are usedextensively. The following definitions are provided to facilitateunderstanding of the invention.

[0093] A structural gene is a DNA sequence that is transcribed intomessenger RNA (mRNA) which is then translated into a sequence of aminoacids characteristic of a specific polypeptide.

[0094] A promoter is a DNA sequence that directs the transcription of astructural gene. Typically, a promoter is located in the 5′ region of agene, proximal to the transcriptional start site of a structural gene.If a promoter is an inducible promoter, then the rate of transcriptionincreases in response to an inducing agent. In contrast, the rate oftranscription is not regulated by an inducing agent if the promoter is aconstitutive promoter.

[0095] An isolated DNA molecule is a fragment of DNA that is notintegrated in the genomic DNA of an organism. For example, a cloned Tcell receptor gene is a DNA fragment that has been separated from thegenomic DNA of a mammalian cell. Another example of an isolated DNAmolecule is a chemically-synthesized DNA molecule that is not integratedin the genomic DNA of an organism.

[0096] An enhancer is a DNA regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

[0097] Complementary DNA (cDNA) is a single-stranded DNA molecule thatis formed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand.

[0098] The term expression refers to the biosynthesis of a gene product.For example, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

[0099] A cloning vector is a DNA molecule, such as a plasmid, cosmid, orbacteriophage, that has the capability of replicating autonomously in ahost cell. Cloning vectors typically contain one or a small number ofrestriction endonuclease recognition sites at which foreign DNAsequences can be inserted in a determinable fashion without loss of anessential biological function of the vector, as well as a marker genethat is suitable for use in the identification and selection of cellstransformed with the cloning vector. Marker genes typically includegenes that provide tetracycline resistance or ampicillin resistance.

[0100] An expression vector is a DNA molecule comprising a gene that isexpressed in a host cell. Typically, gene expression is placed under thecontrol of certain regulatory elements, including constitutive orinducible promoters, tissue-specific regulatory elements, and enhancers.Such a gene is said to be “operably linked to” the regulatory elements.

[0101] A recombinant host may be any prokaryotic or eukaryotic cell thatcontains either a cloning vector or expression vector. This term alsoincludes those prokaryotic or eukaryotic cells that have beengenetically engineered to contain the cloned gene(s) in the chromosomeor genome of the host cell.

[0102] A tumor associated antigen is a protein normally not expressed,or expressed at very low levels, by a normal counterpart. Examples oftumor associated antigens include α-fetoprotein and carcinoembryonicantigen (CEA). Many other illustrations of tumor associated antigens areknown to those of skill in the art. See, for example, Urban et al., Ann.Rev. Immunol. 10: 617 (1992).

[0103] As used herein, an infectious agent denotes both microbes andparasites. A “microbe” includes viruses, bacteria, rickettsia,mycoplasma, protozoa, fungi and like microorganisms. A “parasite”denotes infectious, generally microscopic or very small multicellularinvertebrates, or ova or juvenile forms thereof, which are susceptibleto antibody-induced clearance or lytic or phagocytic destruction, suchas malarial parasites, spirochetes, and the like.

[0104] A multidrug transporter Protein is a membrane-associated proteinwhich transports diverse cytotoxic compounds out of a cell in anenergy-dependent manner. Examples of multidrug transporter proteinsinclude P-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA,CmlA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA,DrrB, TlrC, Bmr, TetA, OprK, and the like.

[0105] An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody.

[0106] The term “antibody fragment” also includes any synthetic orgenetically engineered protein that acts like an antibody by binding toa specific antigen to form a complex. For example, antibody fragmentsinclude isolated fragments consisting of the light chain variableregion, “Fv” fragments consisting of the variable regions of the heavyand light chains, recombinant single chain polypeptide molecules inwhich light and heavy variable regions are connected by a peptide linker(“sFv proteins”), and minimal recognition units consisting of the aminoacid residues that mimic the hypervariable region.

[0107] Humanized antibodies are recombinant proteins in which murinecomplementary determining regions of monoclonal antibodies have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

[0108] As used herein, the term antibody component includes both anentire antibody and an antibody fragment.

[0109] As used herein, a diagnostic or therapeutic agent is a moleculeor atom which is conjugated to an antibody moiety to produce a conjugatewhich is useful for diagnosis or for therapy. Examples of diagnostic ortherapeutic agents include drugs, toxins, immunomodulators, chelators,boron compounds, photoactive agents or dyes, radioisotopes, fluorescentagents, paramagnetic ions or molecules and marker moieties.

[0110] An antibody composite is a polyspecific antibody compositioncomprising at least two substantially monospecific antibody components,wherein at least one antibody component binds with an epitope of amultidrug transporter protein, and wherein at least one antibodycomponent binds with an antigen that is associated with either a tumoror an infectious agent.

[0111] A polyspecific immunoconjugate is a conjugate of an antibodycomposite with a diagnostic or therapeutic agent.

[0112] 2. Production of Rodent Monoclonal Antibodies, HumanizedAntibodies, Primate Antibodies and Human Antibodies

[0113] An antibody composite of the present invention may be derivedfrom a rodent monoclonal antibody (MAb) Rodent monoclonal antibodies tospecific antigens may be obtained by methods known to those skilled inthe art. See, for example, Kohler and Milstein, Nature 256: 495 (1975),and Coligan et al. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1,pages 2.5.1-2.6.7 (John Wiley & Sons 1991) [hereinafter “Coligan”].Briefly, monoclonal antibodies can be obtained by injecting mice with acomposition comprising an antigen, verifying the presence of antibodyproduction by removing a serum sample, removing the spleen to obtainB-lymphocytes, fusing the B-lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones whichproduce antibodies to the antigen, culturing the clones that produceantibodies to the antigen, and isolating the antibodies from thehybridoma cultures.

[0114] MAbs can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

[0115] A wide variety of monoclonal antibodies against tumor associatedantigens or infectious agents have been developed. See, for example,Goldenberg et al., international application publication No. WO 91/11465(1991), Hansen et al., international application publication No. Wo93/23062, and Goldenberg, international application publication No. WO94/04702 (1994), each of which is incorporated by reference in itsentirety.

[0116] Furthermore, such antibodies are readily available fromcommercial sources. For example, rodent monoclonal antibodies that bindwith adenocarcinoma-associated antigen (Cat. No. 121730), humanchorionic gonadotropin (Cat. No. 230740), carcinoembryonic antigen (Cat.Nos. 215920 and 215922), human alpha-fetoprotein (Cat. No. 341646), andthe like can be obtained from Calbiochem-Novabiochem Corp. (San Diego,Calif.). Moreover, rodent monoclonal antibodies that bind with antigenicdeterminants of infectious agents such as Escherichia coli (HB 8178),Legionella pneumophila (CRL 1770), Schistosoma mansoni (HB 8088),Streptococcus, Group A (HB 9696), Treponema pallidum (HB 8134),hepatitis B (CRL 8017), herpes simplex (HB 8181), human immunodeficiencyvirus (HB 9101), among others, can be obtained from American TypeCulture Collection (Rockville, Md.). Furthermore, murine monoclonalantibodies against merozoites and sporozoites of Plasmodium falciparumcan be prepared as described by Goldenberg, U.S. Pat. No. 5,332,567(1994), which is incorporated by reference.

[0117] Methods for producing P-glycoprotein antibodies are well-known tothose of skill in the art. See, for example, Lathan et al., Cancer Res.45: 5064 (1985); Kartner et al., Nature 316: 820 (1985); Hamada et al.,Proc. Nat'l Acad. Sci 83: 7785 (1986); Scheper et al., Int. J. Cancer42: 389 (1988); Rittmann-Grauer et al., Cancer Res. 52: 1810 (1992);Ling et al., U.S. Pat. No. 4,837,306 (1989); Ring et al., internationalpublication No. WO 92/08802; Grauer et al., international publicationNo. WO 93/02105; and Mechetner et al., international publication No. WO93/19094, which are incorporated by reference. Since P-glycoproteinretains its structural identity across different mammalian species(Rubin, U.S. Pat. No. 5,005,588; Kane et al., J. Bioenergetics andBiomembranes 22: 593 (1990)), antibodies raised against P-glycoproteinfrom non-human cells can be used for diagnosis and therapy in humans.Conversely, antibodies raised against human P-glycoprotein should besuitable for veterinary uses.

[0118] Preferred P-glycoprotein antibodies bind with the extracellulardomain of P-glycoprotein, and can be produced against cells that expressthe MDR phenotype as described, for example, by Mechetner et al., supra,and Rittmann-Grauer et al., supra. Alternatively, such antibodies can beobtained using peptides that contain an extracellular epitopeP-glycoprotein. See, for example, Cianfriglia et al., internationalpublication No. WO 93/25700, which is incorporated by reference.

[0119] Those of skill in the art can readily apply standard techniquesto produce antibodies against multidrug transporter proteins ofinfectious agents. Suitable antigens include multidrug transporterproteins such as Bmr, TetA, EmrB, OprK, Smr, and the like. See, forexample, Nikaido et al., supra; Poole et al., J. Bacteriol. 175: 7363(1993); and Childs et al., “The MDR Superfamily of Genes and ItsBiological Implications,” in IMPORTANT ADVANCES IN ONCOLOGY 1994, DeVitaet al., (eds.), pages 21-36 (J.B. Lippincott Co. 1994), which areincorporated by reference. One approach for preparing antibodies againstinfectious agent multidrug transporter proteins is illustrated inExample 6.

[0120] An antibody composite of the present invention may also bederived from a subhuman primate antibody. General techniques for raisingtherapeutically useful antibodies in baboons may be found, for example,in Goldenberg et al., international patent publication No. WO 91/11465(1991), and in Losman et al., Int. J. Cancer 46: 310 (1990), which isincorporated by reference.

[0121] Alternatively, an antibody composite may be derived from a“humanized” monoclonal antibody. Humanized monoclonal antibodies areproduced by transferring mouse complementary determining regions fromheavy and light variable chains of the mouse immunoglobulin into a humanvariable domain, and then, substituting human residues in the frameworkregions of the murine counterparts. The use of antibody componentsderived from humanized monoclonal antibodies obviates potential problemsassociated with the immunogenicity of murine constant regions. Generaltechniques for cloning murine immunoglobulin variable domains aredescribed, for example, by the publication of Orlandi et al., Proc.Nat'l Acad. Sci. USA 86: 3833 (1989), which is incorporated by referencein its entirety., Techniques for producing humanized MAbs are described,for example, by Jones et al., Nature 321: 522 (1986), Riechmann et al.,Nature 332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988),Carter et al., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit.Rev. Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844(1993), each of which is hereby incorporated by reference.

[0122] As an alternative, an antibody composite of the present inventionmay be derived from human antibody fragments isolated from acombinatorial immunoglobulin library. See, for example, Barbas et al.,METHODS: A Companion to Methods in Enzymology 2: 119 (1991), and Winteret al., Ann. Rev. Immunol. 12: 433 (1994), which are incorporated byreference. Cloning and expression vectors that are useful for producinga human immunoglobulin phage library can be obtained, for example, fromSTRATAGENE Cloning Systems (La Jolla, Calif.).

[0123] In addition, an antibody composite of the present invention maybe derived from a human monoclonal antibody. Such antibodies areobtained from transgenic mice that have been “engineered” to producespecific human antibodies in response to antigenic challenge. In thistechnique, elements of the human heavy and light chain locus areintroduced into strains of mice derived from embryonic stem cell linesthat contain targeted disruptions of the endogenous heavy chain andlight chain loci. The transgenic mice can synthesize human antibodiesspecific for human antigens, and the mice can be used to produce humanantibody-secreting hybridomas. Methods for obtaining human antibodiesfrom transgenic mice are described by Green et al., Nature Genet. 7: 13(1994), Lonberg et al., Nature 368: 856 (1994), and Taylor et al., Int.Immun. 6: 579 (1994), which are incorporated by reference.

[0124] 3. Production of Antibody Fragments

[0125] The present invention contemplates the use of antibody fragmentsto produce antibody composites. Antibody fragments can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofthe DNA coding for the fragment. Antibody fragments can be obtained bypepsin or papain digestion of whole antibodies by conventional methods.For example, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab fragments and an Fc fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647 and references contained therein, which patents areincorporated herein in their entireties by reference. Also, see Nisonoffet al., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. J. 73:119 (1959), Edelman et al., in METHODS IN ENZYMOLOGY VOL. 1, page 422(Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and2.10.-2.10.4.

[0126] Other methods of cleaving antibodies, such as separation of heavychains to form monovalent light-heavy chain fragments, further cleavageof fragments, or other enzymatic, chemical or genetic techniques mayalso be used, so long as the fragments bind to the antigen that isrecognized by the intact antibody.

[0127] For example, Fv fragments comprise an association of V_(H) andV_(L) chains. This association can be noncovalent, as described in Inbaret al., Proc. Nat'l Acad. Sci. USA 69: 2659 (1972). Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde. See, for example,Sandhu, supra.

[0128] Preferably, the Fv fragments comprise V_(H) and V_(L) chainswhich are connected by a peptide linker. These single-chain antigenbinding proteins (sFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains which areconnected by an oligonucleotide. The structural gene is inserted into anexpression vector which is subsequently introduced into a host cell,such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow etal., Methods: A Companion to Methods in Enzymology 2: 97 (1991). Alsosee Bird et al., Science 242:423-426 (1988), Ladner et al., U.S. Pat.No. 4,946,778, Pack et al., Bio/Technology 11:1271-1277 (1993), andSandhu, supra.

[0129] Another form of an antibody fragment is a peptide coding for asingle complementarityγ-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick et al.,Methods: A Companion to Methods in Enzymology 2: 106 (1991).

[0130] 4. Production of Antibody composites

[0131] Antibody composites can be prepared by a variety of conventionalprocedures, ranging from glutaraldehyde linkage to more specificlinkages between functional groups. The antibodies and/or antibodyfragments are preferably covalently bound to one another, directly orthrough a linker moiety, through one or more functional groups on theantibody or fragment, e.g., amine, carboxyl, phenyl, thiol, or hydroxylgroups. Various conventional linkers in addition to glutaraldehyde canbe used, e.g., disiocyanates, diiosothiocyanates,bis(hydroxysuccinimide) esters, carbodiimides,maleimidehydroxysuccinimde esters, and the like. The optimal length ofthe linker may vary according to the type of target cell. The mostefficacious linker size can be determined by using antibody compositeswith various linker lengths for the immunochemical staining of a patienttissue sample that contains cells expressing a multidrug transporterprotein and the target antigen. Immunochemical techniques are describedbelow.

[0132] A simple method to produce antibody composites is to mix theantibodies or fragments in the presence of glutaraldehyde to form anantibody composite. The initial Schiff base linkages can be stabilized,e.g., by borohydride reduction to secondary amines. A diiosothiocyanateor carbodiimide can be used in place of glutaraldehyde as anon-site-specific linker.

[0133] The simplest form of an antibody composite is a bispecificantibody comprising binding moieties for a multidrug transporter proteinand an antigen that is associated with a tumor cell or infectious agent.Bispecific antibodies can be made by a variety of conventional methods,e.g., disulfide cleavage and reformation of mixtures of whole IgG or,preferably F(ab′)₂ fragments, fusions of more than one hybridoma to formpolyomas that produce antibodies having more than one specificity, andby genetic engineering. Bispecific antibody composites have beenprepared by oxidative cleavage of Fab′ fragments resulting fromreductive cleavage of different antibodies. This is advantageouslycarried out by mixing two different F(ab′)₂ fragments produced by pepsindigestion of two different antibodies, reductive cleavage to form amixture of Fab′ fragments, followed by oxidative reformation of thedisulfide linkages to produce a mixture of F(ab′)₂ fragments includingbispecific antibody composites containing a Fab′ potion specific to eachof the original epitopes. General techniques for the preparation ofantibody composites may be found, for example, in Nisonhoff et al., ArchBiochem. Biophys. 93: 470 (1961), Hammerling et al., J. Exp. Med. 128:1461 (1968), and U.S. Pat. No. 4,331,647.

[0134] More selective linkage can be achieved by using aheterobifunctional linker such as maleimide-hydroxysuccinimide ester.Reaction of the ester with an antibody or fragment will derivatize aminegroups on the antibody or fragment, and the derivative can then bereacted with, e.g., an antibody Fab fragment having free sulfhydrylgroups (or, a larger fragment or intact antibody with sulfhydryl groupsappended thereto by, e.g., Traut's Reagent). Such a linker is lesslikely to crosslink groups in the same antibody and improves theselectivity of the linkage.

[0135] It is advantageous to link the antibodies or fragments at sitesremote from the antigen binding sites. This can be accomplished by,e.g., linkage to cleaved interchain sulfydryl groups, as noted above.Another method involves reacting an antibody having an oxidizedcarbohydrate portion with another antibody which has at lease one freeamine function. This results in an initial Schiff base (imine) linkage,which is preferably stabilized by reduction to a secondary amine, e.g.,by borohydride reduction, to form the final composite. Suchsite-specific linkages are disclosed, for small molecules, in U.S. Pat.No. 4,671,958, and for larger addends in U.S. Pat. No. 4,699,784.

[0136] In the present context, a bispecific antibody comprises bindingmoieties for a multidrug transporter protein and an antigen that isassociated with a tumor cell or infectious agent. For example, themultidrug transporter protein-binding moiety can be derived fromanti-multidrug transporter protein Mab, while a carcinoembryonic antigen(CEA) binding moiety can be derived from a Class III Mab. Methods forpreparing multidrug transporter protein Mab are described above, whilemethods for preparing Class III anti-CEA Mab are described by Primus etal., Cancer Research 43: 686 (1983), and by Primus et al., U.S. Pat. No.4,818,709, which are incorporated by reference.

[0137] For example, a bispecific antibody can be prepared by obtainingan F(ab′)₂ fragment from an anti-CEA Class III Mab, using the techniquesdescribed above. The interchain disulfide bridges of the anti-CEA ClassIII F(ab′)₂ fragment are gently reduced with cysteine, taking care toavoid light-heavy chain linkage, to form Fab′-SH fragments. The SHgroup(s) is(are) activated with an excess of bis-maleimide linker(1,1′-(methylenedi-4,1-phenylene)bis-malemide). The multidrugtransporter protein Mab is converted to Fab′-SH and then reacted withthe activated anti-CEA Class III Fab′-SH fragment to obtain a bispecificantibody.

[0138] Alternatively, such bispecific antibodies can be produced byfusing two hybridoma cell lines that produce anti-multidrug transporterprotein Mab and anti-CEA Class III Mab. Techniques for producingtetradomas are described, for example, by Milstein et al., Nature 305:537 (1983) and Pohl et al., Int. J. Cancer 54: 418 (1993).

[0139] Finally, such bispecific antibodies can be produced by geneticengineering. For example, plasmids containing DNA coding for variabledomains of an anti-CEA Class III Mab can be introduced into hybridomasthat secrete anti-multidrug transporter protein antibodies. Theresulting “transfectomas” produce bispecific antibodies that bind CEAand the multidrug transporter protein. Alternatively, chimeric genes canbe designed that encode both anti-multidrug transporter protein, andanti-CEA binding domains. General techniques for producing bispecificantibodies by genetic engineering are described, for example, bySongsivilai et al., Biochem. Biophys. Res. Commun. 164: 271 (1989);Traunecker et al., EMBO J. 10: 3655 (1991); and Weiner et al., J.Immunol. 147: 4035 (1991)

[0140] A polyspecific antibody composite can be obtained by addingvarious antibody components to a bispecific antibody composite. Forexample, a bispecific antibody can be reacted with 2-iminothiolane tointroduce one or more sulfhydryl groups for use in coupling thebispecific antibody to an antibody component that binds an epitope of amultidrug transporter protein that is distinct from the epitope bound bythe bispecific antibody, using the bis-maleimide activation proceduredescribed above. These techniques for producing antibody composites arewell known to those of skill in the art. See, for example, U.S. Pat. No.4,925,648, and Goldenberg, international publication No. WO 92/19273,which are incorporated by reference.

[0141] 5. Preparation of Polyspecific Immunoconjugates

[0142] Polyspecific immunoconjugates can be prepared by indirectlyconjugating a diagnostic or therapeutic agent to an antibody composite.General techniques are described in Shih et al., Int. J. Cancer41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106 (1990); andShih et al., U.S. Pat. No. 5,057,313. The general method involvesreacting an antibody component having an oxidized carbohydrate portionwith a carrier polymer that has at least one free amine function andthat is loaded with a plurality of drug, toxin, chelator, boron addends,or other diagnostic or therapeutic agent. This reaction results in aninitial Schiff base (imine) linkage, which can be stabilized byreduction to a secondary amine to form the final conjugate.

[0143] The carrier polymer is preferably an aminodextran or polypeptideof at least 50 amino acid residues, although other substantiallyequivalent polymer carriers can also be used. Preferably, the finalpolyspecific immunoconjugate is soluble in an aqueous solution, such asmammalian serum, for ease of administration and effective targeting foruse in diagnosis or therapy. Thus, solubilizing functions on the carrierpolymer will enhance the serum solubility of the final polyspecificimmunoconjugate. Solubilizing functions also are important for use ofpolyspecific immunoconjugates for immunochemical detection, as describedbelow. In particular, an aminodextran will be preferred.

[0144] The process for preparing a polyspecific immunoconjugate with anaminodextran carrier typically begins with a dextran polymer,advantageously a dextran of average molecular weight of about10,000-100,000. The dextran is reacted with an oxidizing agent to effecta controlled oxidation of a portion of its carbohydrate rings togenerate aldehyde groups. The oxidation is conveniently effected withglycolytic chemical reagents such as NaIO₄, according to conventionalprocedures.

[0145] The oxidized dextran is then reacted with a polyamine, preferablya diamine, and more preferably, a mono- or polyhydroxy diamine. Suitableamines include ethylene diamine, propylene diamine, or other likepolymethylene diamines, diethylene triamine or like polyamines,1,3-diamino-2-hydroxypropane, or other like hydroxylated diamines orpolyamines, and the like. An excess of the amine relative to thealdehyde groups of the dextran is used to insure substantially completeconversion of the aldehyde functions to Schiff base groups.

[0146] A reducing agent, such as NaBH₄, NaBH₃CN or the like, is used toeffect reductive stabilization of the resultant Schiff baseintermediate. The resultant adduct can be purified by passage through aconventional sizing column to remove cross-linked dextrans.

[0147] Other conventional methods of derivatizing a dextran to introduceamine functions can also be used, e.g., reaction with cyanogen bromide,followed by reaction with a diamine.

[0148] The aminodextran is then reacted with a derivative of theparticular drug, toxin, chelator, paramagnetic ion, boron addend, orother diagnostic or therapeutic agent to be loaded, in an activatedform, preferably, a carboxyl-activated derivative, prepared byconventional means, e.g., using dicyclohexylcarbodiimide (DCC) or awater soluble variant thereof, to form an intermediate adduct.

[0149] Alternatively, polypeptide toxins such as pokeweed antiviralprotein or ricin A-chain, and the like, can be coupled to aminodextranby glutaraldehyde condensation or by reaction of activated carboxylgroups on the protein with amines on the aminodextran.

[0150] Chelators for radiometals or magnetic resonance enhancers arewell-known in the art. Typical are derivatives ofethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaaceticacid (DTPA). These chelators typically have groups on the side chain bywhich the chelator can be attached to a carrier. Such groups include,e.g., benzylisothiocyanate, by which the DTPA or EDTA can be coupled tothe amine group of a carrier. Alternatively, carboxyl groups or aminegroups on a chelator can be coupled to a carrier by activation or priorderivatization and then coupling, all by well-known means.

[0151] Labels such as enzymes, fluorescent compounds, electron transferagents, and the like can be linked to a carrier by conventional methodswell known to the art. These labeled carriers and the polyspecificimmunoconjugates prepared from them can be used for immunochemicaldetection, as described below.

[0152] Boron addends, such as carboranes, can be attached to antibodycomponents by conventional methods. For example, carboranes can beprepared with carboxyl functions on pendant side chains, as is wellknown in the art. Attachment of such carboranes to a carrier, e.g.,aminodextran, can be achieved by activation of the carboxyl groups ofthe carboranes and condensation with amines on the carrier to produce anintermediate conjugate. Such intermediate conjugates are then attachedto. antibody components to produce therapeutically useful polyspecificimmunoconjugates, as described below.

[0153] A polypeptide carrier can be used instead of aminodextran, butthe polypeptide carrier must have at least 50 amino acid residues in thechain, preferably 100-5000 amino acid residues. At least some of theamino acids should be lysine residues or glutamate or aspartateresidues. The pendant amines of lysine residues and pendant carboxylatesof glutamine and aspartate are convenient for attaching a drug, toxin,chelator, boron addend or other diagnostic or therapeutic agent.Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, copolymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andpolyspecific immunoconjugate.

[0154] Conjugation of the intermediate conjugate with the antibodycomponent is effected by oxidizing the carbohydrate portion of theantibody component and reacting the resulting aldehyde (and ketone)carbonyls with amine groups remaining on the carrier after loading witha drug, toxin, chelator, boron addend, or other diagnostic ortherapeutic agent. Alternatively, an intermediate conjugate can beattached to an oxidized antibody component via amine groups that havebeen introduced in the intermediate conjugate after loading with thediagnostic or therapeutic agent. Oxidation is conveniently effectedeither chemically, e.g., with NaIO₄ or other glycolytic reagent, orenzymatically, e.g., with neuraminidase and galactose oxidase. In thecase of an aminodextran carrier, not all of the amines of theaminodextran are typically used for loading a diagnostic or therapeuticagent. The remaining amines of aminodextran condense with the oxidizedantibody component to form Schiff base adducts, which are thenreductively stabilized, normally with a borohydride reducing agent.

[0155] Analogous procedures are used to produce other polyspecificimmunoconjugates according to the invention. Loaded polypeptide carrierspreferably have free lysine residues remaining for condensation with theoxidized carbohydrate portion of an antibody component. Carboxyls on thepolypeptide carrier can, if necessary, be converted to amines by, e.g.,activation with DCC and reaction with an excess of a diamine.

[0156] The final polyspecific immunoconjugate is purified usingconventional techniques, such as sizing chromatography on SephacrylS-300.

[0157] Alternatively, polyspecific immunoconjugates can be prepared bydirectly conjugating an antibody component with a diagnostic ortherapeutic agent. The general procedure is analogous to the indirectmethod of conjugation except that a diagnostic or therapeutic agent isdirectly attached to an oxidized antibody component.

[0158] It will be appreciated that other diagnostic or therapeuticagents can be substituted for the chelators described herein. Those ofskill in the art will be able to devise conjugation schemes withoutundue experimentation.

[0159] In addition, those of skill in the art will recognize numerouspossible variations of the conjugation methods. For example, thecarbohydrate moiety can be used to attach polyethyleneglycol in order toextend the half-life of an intact antibody, or antigen-binding fragmentthereof, in blood, lymph, or other extracellular fluids. Moreover, it ispossible to construct a “divalent immunoconjugate” by attaching adiagnostic or therapeutic agent to a carbohydrate moiety and to a freesulfhydryl group. Such a free sulfhydryl group may be located in thehinge region of the antibody component.

[0160] 6. Use of Polyspecific Immunoconjugates and Antibody Compositesfor Diagnosis

[0161] A. In Vitro Diagnosis

[0162] The present invention contemplates the use of polyspecificimmunoconjugates and antibody composites to screen biological samples invitro for the expression of P-glycoprotein by tumor cells. For example,the polyspecific immunoconjugates and antibody composites of the presentinvention can be used to detect the presence of P-glycoprotein and tumorassociated antigen in tissue sections prepared from a biopsy specimen.Such immunochemical detection can be used to determine the abundance ofP-glycoprotein and to determine the distribution of P-glycoprotein inthe examined tissue. General immunochemistry techniques are well-knownto those of ordinary skill. See, for example, Ponder, “Cell MarkingTechniques and Their Application,” in MAMMALIAN DEVELOPMENT: A PRACTICALAPPROACH, Monk (ed.), pages 115-38 (IRL Press 1987), Volm et al., Eur.J. Cancer Clin. Oncol. 25: 743 (1989), Coligan at pages 5.8.1-5.8.8, andAusubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages14.6.1 to 14.6.13 (Wiley Interscience 1990). Also, see generally, Manson(ed.), METHODS IN MOLECULAR BIOLOGY, VOL.10: IMMUNOCHEMICAL PROTOCOLS(The Humana Press, Inc. 1992). Moreover, methods for the immunochemicaldetection of P-glycoprotein are described, for example, by Dalton etal., Blood 73: 747 (1989), and Volm et al., Eur. J. Cancer Clin. Oncol.25: 743 (1989).

[0163] In addition, the present invention contemplates the use ofpolyspecific immunoconjugates and antibody composites to screenbiological samples in vitro for the expression of a multidrugtransporter protein by an infectious agent. For example, thepolyspecific immunoconjugates and antibody composites of the presentinvention can be used to detect the presence of OprK protein in clinicalisolates. The presence of this particular multidrug transporter proteinwould indicate that the tissue was infected with multidrug resistantPsuedomonas aeruginosa.

[0164] Moreover, immunochemical detection techniques can be used tooptimize antibody composites for subsequent in vivo diagnosis andtherapy in the form of antibody composites per se or as polyspecificimmunoconjugates. Accordingly, immunochemical detection can be performedwith a battery of antibody composites to identify the most appropriatecombination of antibody components for subsequent in vivo diagnosis andtherapy. For example, an antibody moiety that binds the c-erb B2proto-oncogene product may be more suitable for a particular breastcancer than an antibody moiety that binds carcinoembryonic antigen.After a suitable combination of antibody components have beenidentified, further in vitro testing can be used to delineate the mostefficacious linker size in the antibody composite, as discussed above.

[0165] Immunochemical detection can be performed by contacting abiological sample with an antibody composite and then contacting thebiological sample with a detectably labeled molecule which binds to theantibody composite. For example, the detectably labeled molecule cancomprise an antibody moiety that binds the antibody composite.Alternatively, the antibody composite can be conjugated withavidin/streptavidin (or biotin) and the detectably labeled molecule cancomprise biotin (or avidin/streptavidin). Numerous variations of thisbasic technique are well-known to those of skill in the art.

[0166] Alternatively, an antibody composite can be conjugated with adiagnostic agent to form a polyspecific immunoconjugate. Antibodycomposites can be detectably labeled with any appropriate marker moiety,for example, a radioisotope, a fluorescent label, a chemiluminescentlabel, an enzyme label, a bioluminescent label or colloidal gold.Methods of making and detecting such detectably-labeled polyspecificimmunoconjugates are well-known to those of ordinary skill in the art,and are described in more detail below.

[0167] The marker moiety can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C .

[0168] Polyspecific immunoconjugates also can be labeled with afluorescent compound. The presence of a fluorescently-labeled antibodycomponent is determined by exposing the polyspecific immunoconjugate tolight of the proper wavelength and detecting the resultant fluorescence.Fluorescent labeling compounds include fluorescein isothiocyanate,rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine.

[0169] Alternatively, polyspecific immunoconjugates can be detectablylabeled by coupling an antibody component to a chemiluminescentcompound. The presence of the chemiluminescent-tagged polyspecificimmunoconjugate is determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofchemiluminescent labeling compounds include luminol, isoluminol, anaromatic acridinium ester, an imidazole, an acridinium salt and anoxalate ester.

[0170] Similarly, a bioluminescent compound can be used to labelpolyspecific immunoconjugates of the present invention. Bioluminescenceis a type of chemiluminescence found in biological systems in which acatalytic protein increases the efficiency of the chemiluminescentreaction. The presence of a bioluminescent protein is determined bydetecting the presence of luminescence. Bioluminescent compounds thatare useful for labeling include luciferin, luciferase and aequorin.

[0171] Alternatively, polyspecific immunoconjugates can be detectablylabeled by linking an antibody component to an enzyme. When thepolyspecific immunoconjugates-enzyme conjugate is incubated in thepresence of the appropriate substrate, the enzyme moiety reacts with thesubstrate to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorometric or visual means. Examplesof enzymes that can be used to detectably label polyspecificimmunoconjugates include β-galactosidase, glucose oxidase, peroxidaseand alkaline phosphatase.

[0172] Those of skill in the art will know of other suitable labelswhich can be employed in accordance with the present invention. Thebinding of marker moieties to antibody components can be accomplishedusing standard techniques known to the art. Typical methodology in thisregard is described by Kennedy et al., Clin. Chim. Acta 70: 1 (1976),Schurs et al., Clin. Chim. Acta 81: 1 (1977), Shih et al., Int'l J.Cancer 46: 1101 (1990), Stein et al., Cancer Res. 50: 1330 (1990),supra, and Stein et al., Int. J. Cancer 55: 938 (1993). Also, seegenerally, Coligan.

[0173] In addition, the convenience and versatility of immunochemicaldetection can be enhanced by using antibody components that have beenconjugated with avidin, streptavidin, and biotin. See, for example,Wilchek et al. (eds.), Avidin-Biotin Technology, METHODS IN ENZYMOLOGY,VOL. 184 (Academic Press 1990), and Bayer et al., “ImmunochemicalApplications of Avidin-Biotin Technology,” in METHODS IN MOLECULARBIOLOGY, VOL. 10, Manson (ed.), pages 149-162 (The Human Press, Inc.1992).

[0174] Thus, the above-described immunochemical detection methods can beused to assist in the diagnosis or staging of a pathological condition.These techniques also can be used to identify the most suitablecomposition of antibody composite or polyspecific immunoconjugate forsubsequent in vivo diagnosis and therapy.

[0175] B. In Vivo Diagnosis

[0176] The present invention also contemplates the use of antibodycomposites and polyspecific immunoconjugates for in vivo diagnosis. Themethod of diagnostic imaging with radiolabeled MAbs is well-known. Inthe technique of immunoscintigraphy, for example, antibodies are labeledwith a gamma-emitting radioisotope and introduced into a patient. Agamma camera is used to detect the location and distribution ofgamma-emitting radioisotopes. See, for example, Srivastava (ed.),RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY (Plenum Press1988), Chase, “Medical Applications of Radioisotopes,” in REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition, Gennaro et al. (eds.), pp.624-652 (Mack Publishing Co., 1990), Brown, “Clinical Use of MonoclonalAntibodies,” in BIOTECHNOLOGY AND PHARMACY 227-49, Pezzuto et al. (eds.)(Chapman & Hall 1993), and Goldenberg, Calif.—A Cancer Journal forClinicians 44: 43 (1994). For diagnostic imaging, radioisotopes may bebound to an antibody composite either directly, or indirectly by usingan intermediary functional group. Useful intermediary functional groupsinclude chelators such as ethylenediaminetetraacetic acid anddiethylenetriaminepentaacetic acid. For example, see Shih et al., supra,and U.S. Pat. No. 5,057,313. Also, see Griffiths, U.S. Pat. No.5,128,119 (1992).

[0177] The radiation dose delivered to the patient is maintained at aslow a level as possible through the choice of isotope for the bestcombination of minimum half-life, minimum retention in the body, andminimum quantity of isotope which will permit detection and accuratemeasurement. Examples of radioisotopes that can be bound to antibodycomposites and are appropriate for 30, diagnostic imaging includeγ-emitters and positron-emitters such as ^(99m)Tc, ⁶⁷Ga, ¹¹¹In, ¹²³I,¹²⁴I, ¹²⁵I, ¹³¹I, ⁵¹Cr, ⁸⁹Zr, ¹⁸F and ⁶⁸Ga. Other suitable radioisotopesare known to those of skill in the art.

[0178] Preferred γ-emitters have a gamma radiation emission peak in therange of 50-500 Kev, primarily because the state of the art forradiation detectors currently favors such labels. Examples of suchγ-emitters include ^(99m)Tc, ⁶⁷Ga, ¹²³I, ¹²⁵I and ¹³¹I.

[0179] Antibody composites also can be labeled with paramagnetic ionsfor purposes of in vivo diagnosis. Elements that are particularly usefulfor magnetic resonance imaging include Gd, Mn, Dy and Fe ions.

[0180] A high background level of non-targeted antibody provides a majorimpediment to in vivo diagnosis methodology. However, the ratio oftarget to nontarget radiolabeled antibody can be enhanced through theuse of a nonlabeled second antibody which scavenges and promotes theclearance of the nontargeted circulating radiolabeled antibody. Thesecond antibody may be whole IgG or IgM, or a fragment of IgG or IgM, solong as it is capable of binding the radiolabeled antibody to form acomplex which is cleared from the circulation and nontarget spaces morerapidly than the radiolabeled antibody alone. In the present context,suitable second antibodies may bind with either the Fc portion orvariable region of a radiolabeled polyspecific immunoconjugate. See, forexample, Goldenberg, U.S. Pat. No. 4,624,846, Goldenberg, internationalpublication No. WO 92/19273, and Sharkey et al., Int. J. Cancer 51: 266(1992), which are incorporated by reference.

[0181] For example, the location of multidrug resistant (MDR) tumorcells, MDR HIV-infected cells or MDR infectious agents in a mammalhaving a multidrug resistant disease caused by a tumor or infectiousagent can be determined by parenterally injecting the mammal with apolyspecific immunoconjugate comprising (1) at least one antibodycomponent that binds with a first epitope of a multidrug transporterprotein, (2) at least one antibody component that binds with a firstepitope of an antigen that is associated with the tumor or infectiousagent, and (3) a diagnostic agent. Subsequently, the mammal is injectedwith an antibody or antibody fragment that binds with the polyspecificimmunoconjugate in an amount that is sufficient to decrease the level ofcirculating polyspecific immunoconjugate by about 10-85% within 2 to 72hours. The mammal is then scanned with a detector to locate the site orsites of uptake of the polyspecific immunoconjugate. See Goldenberg,U.S. Pat. No. 4,624,846.

[0182] In an alternate approach, detection methods are improved bytaking advantage of the binding between avidin/streptavidin and biotin.Avidin, found in egg whites, has a very high binding affinity forbiotin, which is a B-complex vitamin. Streptavidin, isolated fromStreptomyces avidinii, is similar to avidin, but has lower non-specifictissue binding and therefore, streptavidin often is used in place ofavidin. A basic diagnostic method comprises administering an antibodycomposite conjugated with avidin/streptavidin (or biotin), injecting aclearing composition comprising biotin (or avidin/streptavidin), andadministering a conjugate of a diagnostic agent and biotin (oravidin/streptavidin). Preferably, the biotin (or avidin/streptavidin)component of the clearing composition is coupled with a carbohydratemoiety (such as dextran) or a polyol group (e.g., polyethylene glycol)to decrease immunogenicity and permit repeated applications.

[0183] A modification of the basic method is performed by parenterallyinjecting a mammal with an antibody composite which has been conjugatedwith avidin/streptavidin (or biotin), injecting a clearing compositioncomprising biotin (or avidin/streptavidin), and parenterally injecting apolyspecific immunoconjugate according to the present invention, whichfurther comprises avidin/streptavidin (or biotin). See Goldenberg,international publication No. WO 94/04702, which is incorporated byreference.

[0184] In a further variation of this method, improved detection can beachieved by conjugating multiple avidin/streptavidin or biotin moietiesto a polymer which, in turn, is conjugated to an antibody component.Adapted to the present invention, antibody composites or polyspecificimmunoconjugates can be produced which contain multipleavidin/streptavidin or biotin moieties. Techniques for constructing andusing multiavidin/multistreptavidin and/or multibiotin polymerconjugates to obtain amplification of targeting are disclosed byGriffiths, international application No. PCT/US94/04295, which isincorporated by reference.

[0185] In another variation, improved detection is achieved by injectinga targeting antibody composite conjugated to biotin (oravidin/streptavidin), injecting at least one dose of anavidin/streptavidin (or biotin) clearing agent, and injecting adiagnostic composition comprising a conjugate of biotin (oravidin/streptavidin) and a naturally occurring metal atom chelatingprotein which is chelated with a metal detection agent. Suitabletargeting proteins according to the present invention would be ferritin,metallothioneins, ferredoxins, and the like. This approach is disclosedby Goldenberg et al., international application No. PCT/US94/05149,which is incorporated by reference.

[0186] Polyspecific immunoconjugates which comprise a radiolabel alsocan be used to detect multidrug resistant (MDR) tumor cells, MDRHIV-infected cells or MDR infectious agents in the course ofintraoperative and endoscopic examination using a small radiationdetection probe. See Goldenberg U.S. Pat. No. 4,932,412, which isincorporated by reference. As an illustration of the basic approach, asurgical or endoscopy subject is injected parenterally with apolyspecific immunoconjugate comprising (1) at least one antibodycomponent that binds with a first epitope of a multidrug transporterprotein, (2) at least one antibody component that binds with a firstepitope of an antigen that is associated with a tumor or infectiousagent, and (3) a radioisotope. Subsequently, the surgically exposed orendoscopically accessed interior of the body cavity of the subject isscanned at close range with a radiation detection probe to detect thesites of accretion of the polyspecific immunoconjugate.

[0187] In a variation of this method, a photoactive agent or dye, suchas dihematoporphyrin ether (Photofrin II), is injected systemically andsites of accretion of the agent or dye are detected by laser-inducedfluorescence and endoscopic imaging. See Goldenberg, internationalapplication No. PCT/US93/04098, which is incorporated by reference. Theprior art discloses imaging techniques using certain dyes that areaccreted by lesions, such as tumors, and which are in turn activated bya specific frequency of light. These methods are described, for example,in Dougherty et al., Cancer Res. 38: 2628 (1978); Dougherty, Photochem.Photobiol. 45: 879 (1987); Doiron et al. (eds.), PORPHYRIN LOCALIZATIONAND TREATMENT OF TUMORS (Alan Liss, 1984); and van den Bergh, Chem.Britain 22: 430 (1986), which are incorporated herein in their entiretyby reference.

[0188] In a basic technique, a subject is injected parenterally with apolyspecific immunoconjugate comprising (1) at least one antibodycomponent that binds with a first epitope of a multidrug transporterprotein, (2) at least one antibody component that binds with a firstepitope of an antigen that is associated with a tumor or infectiousagent, and (3) a photoactive agent or dye. Sites of accretion aredetected using a light source provided by an endoscope or during asurgical procedure.

[0189] The detection of polyspecific immunoconjugate duringintraoperative or endoscopic examination can be enhanced through the useof second antibody or avidin/streptavidin/biotin clearing agents, asdiscussed above.

[0190] In these endoscopic techniques the detection means can beinserted into a body cavity through an orifice, such as, the mouth,nose, ear, anus, vagina or incision. As used herein, the term“endoscope” is used generically to refer to any scope introduced into abody cavity, e.g., an anally introduced endoscope, an orally introducedbronchoscope, a urethrally introduced cystoscope, an abdominallyintroduced laparoscope or the like. Certain of these may benefit greatlyfrom further progress in miniaturization of components and their utilityto practice the method of the present invention will be enhanced as afunction of the development of suitably microminiaturized components forthis type of instrumentation. Highly miniaturized probes which could beintroduced intravascularly, e.g., via catheters or the like, are alsosuitable for use in the embodiments of the invention for localizing MDRtumor cells, MDR HIV-infected cells or MDR infectious agents.

[0191] 7. Use of Polyspecific Immunoconjugates and Antibody Compositesfor Therapy

[0192] The present invention also contemplates the use of antibodycomposites and polyspecific immunoconjugates for immunotherapy. Anobjective of immunotherapy is to deliver cytotoxic doses ofradioactivity, toxin, or drug to target cells, while minimizing exposureto non-target tissues. The polyspecific immunoconjugates and antibodycomposites of the present invention are expected to have a greaterbinding specificity than multidrug transporter protein MAbs, since thepolyspecific immunoconjugates and antibody composites comprise moietiesthat bind to at least one multidrug transporter protein epitope and anantigen associated with either a tumor or an infectious agent.

[0193] For example, a therapeutic polyspecific immunoconjugate maycomprise an α-emitting radioisotope, a β-emitting radioisotope, aγ-emitting radioisotope, an Auger electron emitter, a neutron capturingagent that emits α-particles or a radioisotope that decays by electroncapture. Suitable radioisotopes include ¹⁹⁸Au, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y,¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu, ²¹¹At, and the like.

[0194] As discussed above, a radioisotope can be attached to an antibodycomposite directly or indirectly, via a chelating agent. For example,⁶⁷Cu, considered one of the more promising radioisotopes forradioimmunotherapy due to its 61.5 hour half-life and abundant supply ofbeta particles and gamma rays, can be conjugated to an antibodycomposite using the chelating agent,p-bromoacetamido-benzyl-tetraethylaminetetraacetic acid (TETA). Chase,supra. Alternatively, ⁹⁰Y, which emits an energetic beta particle, canbe coupled to an antibody composite using diethylenetriaminepentaaceticacid (DTPA). Moreover, a method for the direct radiolabeling of theantibody composite with ¹³¹I is described by Stein et al., AntibodyImmunoconj. Radiopharm. 4: 703 (1991).

[0195] Alternatively, boron addends such as carboranes can be attachedto antibody composites. Carboranes can be prepared with carboxylfunctions on pendant side chains, as is well-known in the art.Attachment of carboranes to a carrier, such as aminodextran, can beachieved by activation of the carboxyl groups of the carboranes andcondensation with amines on the carrier. The intermediate conjugate isthen conjugated to the antibody composite. After administration of thepolyspecific immunoconjugate, a boron addend is activated by thermalneutron irradiation and converted to radioactive atoms which decay byα-emission to produce highly toxic, short-range effects.

[0196] In addition, therapeutically useful polyspecific immunoconjugatescan be prepared in which an antibody composite is conjugated to a toxinor a chemotherapeutic drug. Illustrative of toxins which are suitablyemployed in the preparation of such conjugates are ricin, abrin, humanribonuclease, pokeweed antiviral protein, gelonin, diphtherin toxin, andPseudomonas endotoxin. See, for example, Pastan et al., Cell 47: 641(1986), and Goldenberg, CA—A Cancer Journal for Clinicians 44: 43(1994). Other suitable toxins are known to those of skill in the art.

[0197] Useful cancer chemotherapeutic drugs for the preparation ofpolyspecific immunoconjugates include nitrogen mustards, alkylsulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidineanalogs, purine analogs, antibiotics, epipodophyllotoxins, platinumcoordination complexes, hormones, and the like. Chemotherapeutic drugsthat are useful for treatment of infectious agents include antiviraldrugs (such as AZT, 2′,3′-dideoxyinosine and 2′,3′-dideoxycytidine),antimalarial drugs (such as chloroquine and its congeners,diaminopyrimidines, mefloquine), antibacterial agents, antifungalagents, antiprotozoal agents, and the like. Suitable chemotherapeuticagents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed.(Mack Publishing Co. 1990), and in GOODMAN AND GILMAN'S THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co.1985), which are incorporated by reference. Other suitablechemotherapeutic agents, such as experimental drugs, are known to thoseof skill in the art.

[0198] In addition, therapeutically useful polyspecific immunoconjugatescan be obtained by conjugating photoactive agents or dyes to an antibodycomposite. Fluorescent and other chromogens, or dyes, such as porphyrinssensitive to visible light, have been used to detect and to treatlesions by directing the suitable light to the lesion (cited above). Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy (Jori et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORSAND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain 22: 430 (1986)). Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy (Mew et al.,J. Immunol. 130: 1473 (1983); idem., Cancer Res. 45: 4380 (1985);Oseroff et al., Proc. Natl. Acad. Sci. USA 83: 8744 (1986); idem.,Photochem. Photobiol. 46: 83 (1987); Hasan et al., Prog. Clin. Biol.Res. 288: 471 (1989); Tatsuta et al., Lasers Surg. Med. 9: 422 (1989);Pelegrin et al., Cancer 67: 2529 (1991)—all incorporated in theirentirety herein by reference). However, these earlier studies did notinclude use of endoscopic therapy applications, especially with the useof antibody fragments or subfragments. Thus, the present inventioncontemplates the therapeutic use of polyspecific immunoconjugatescomprising photoactive agents or dyes. The general methodology isdescribed above in relation to the use of such polyspecificimmunoconjugates for diagnosis.

[0199] Moreover, therapeutically useful polyspecific immunoconjugatescan be prepared in which an antibody composite is conjugated to acompound that reverses multidrug resistance. Such “chemosensitizingagents” include verapamil and its analogs, calmodulin antagonists,anthracycline and Vinca alkaloid analogs, and the like. See, forexample, Endicott et al., Ann. Rev. Biochem. 58: 137 (1989), Ford etal., Pharmacol. Rev. 42: 155 (1990) and Calabresi et al., PPO Updates8:1 (1994). See also Sarkadi et al., FASEB J. 8: 766 (1994), whichprovides methods to identify hydrophobic peptide derivatives thatreverse multidrug resistance. These polyspecific immunoconjugates may beadministered prior to, or concurrent with, the administration ofappropriate chemotherapeutic drugs.

[0200] As an alternative, unconjugated chemosensitizing agents may beadministered with polyspecific immunoconjugates comprising a toxin orchemotherapeutic drug. Typical modes of administration and dosages ofchemosensitizing agents are described, for example, by Presant et al.,Am. J. Clin. Oncol. 9: 355 (1986), Cairo et al., Cancer Res. 49: 1063(1989), Miller et al., J. Clin. Oncol. 9: 37 (1991) and Calabresi etal., supra, Rubin, U.S. Pat. No. 5,005,588, and Levy, U.S. Pat. No.5,258,372, which are incorporated by reference.

[0201] In addition, therapeutic polyspecific immunoconjugates cancomprise an immunomodulator moiety. As used herein, the term“immunomodulator” includes cytokines, stem cell growth factors, tumornecrosis factors (TNF) and hematopoietic factors, such as interleukins(e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-6 and IL-10), colonystimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF)and granulocyte macrophage-colony stimulating factor (GM-CSF)),interferons (e.g., interferons-α, -β and -γ), the stem cell growthfactor designated “S1 factor,” erythropoietin and thrombopoietin.Examples of suitable immunomodulator moieties include IL-2, IL-6, IL-10,interferon-′, TNF-α, and the like.

[0202] Such polyspecific immunoconjugates provide a means to deliver animmunomodulator to a target cell and are particularly useful againsttumor cells and mammalian cells that express an infectious agent antigenon the cell surface, such as HIV-infected cells. The cytotoxic effectsof immunomodulators are well known to those of skill in the art. See,for example, Klegerman et al., “Lymphokines and Monokines,” inBIOTECHNOLOGY AND PHARMACY, Pessuto et al. (eds.), pages 53-70 (Chapman& Hall 1993). As an illustration, type I interferons and interferon-γinduce an antiviral state in various cells by activating2′,5′-oligoadenylate synthetase and protein kinase. Moreover,interferons can inhibit cell proliferation by inducing increasedexpression of class I histocompatibility antigens on the surface ofvarious cells and thus, enhance the rate of destruction of cells bycytotoxic T lymphocytes. Furthermore, tumor necrosis factors, such asTNF-α, are believed to produce cytotoxic effects by inducing DNAfragmentation.

[0203] The present invention also contemplates two-, three- or four-steptargeting strategies to enhance antibody therapy. General techniquesinclude the use of antibody components conjugated with avidin,streptavidin or biotin, and the use of second antibodies that bind withthe primary immunoconjugate, as discussed above. See, for example,Goodwin et al., Eur. J. Nucl. Med. 9:209 (1984), Goldenberg et al., J.Nucl. Med. 28:1604 (1987), Hnatowich et al., J. Nucl. Med. 28: 1294(1987), Paganelli et al., Cancer Res. 51: 5960 (1991), Goldenberg,international publication No. WO 92/19273, Sharkey et al., Int. J.Cancer 51: 266 (1992), and Goldenberg, international application No. WO94/04702, which are incorporated by reference. Also, see Griffiths,international application No. PCT/US94/04295, which describes a methodusing multiavidin and/or multibiotin polymer conjugates, and Goldenberget al., international application No. PCT/US94/05149, which disclosesimproved methods for therapy with chelatable radiometals.

[0204] For example, a mammal having a multidrug resistant disease causedby a tumor or infectious agent may be treated by parenterally injectingthe mammal with a polyspecific immunoconjugate comprising (1) at leastone antibody component that binds with a first epitope of a multidrugtransporter protein, (2) at least one antibody component that binds witha first epitope of an antigen that is associated with the tumor orinfectious agent, and (3) a therapeutic agent. Subsequently, the mammalis injected with an antibody or antibody fragment that binds with thepolyspecific immunoconjugate in an amount that is sufficient to decreasethe level of circulating polyspecific immunoconjugate by about 10-85%within 2 to 72 hours.

[0205] In an alternative approach to enhancing the therapeutic indexcomprises administering an antibody composite conjugated withavidin/streptavidin (or biotin), injecting a clearing compositioncomprising biotin (or avidin/streptavidin), and administering aconjugate of a therapeutic agent and avidin/streptavidin (or biotin), asdiscussed above.

[0206] The present invention also contemplates a method of therapy usingunconjugated antibody composites. Investigators have found thatP-glycoprotein antibodies can restore drug sensitivity in multidrugresistant cultured cells and multidrug resistant human tumor xenograftsin nude mice. Grauer et al., European patent application No. EP-0 569141, Rittmann-Grauer et al., Cancer Res. 52: 1810 (1992), Pearson etal., J. Nat'l Cancer Inst. 83: 1386 (1991), and Iwahashi et al., CancerRes. 53: 5475 (1993). P-glycoprotein antibodies also can inhibit thegrowth of multidrug resistant human xenografts in nude mice. Grauer etal., European patent application No. EP-0 569,141. Accordingly, the morespecific antibody composites of the present invention provide animproved method to treat a mammal having a multidrug resistant diseasecaused by a tumor or infectious agent in which the multidrug resistantcells overexpress P-glycoprotein. Moreover, the antibody composites ofthe present invention can be used to inhibit active drug efflux ininfectious agents and thus, restore sensitivity to chemotherapy.

[0207] Antibody composites may be administered alone, or in conjugationwith the conventional chemotherapeutic agents described above. Modes ofchemotherapeutic administration and suitable dosages are well known tothose of skill in the art. See, for example, REMINGTON'S PHARMACEUTICALSCIENCES, 18th Ed. (Mack Publishing Co. 1990), and GOODMAN AND GILMAN'STHE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan PublishingCo. 1985).

[0208] In general, the dosage of administered polyspecificimmunoconjugates and antibody composites will vary depending upon suchfactors as the patient's age, weight, height, sex, general medicalcondition and previous medical history. Typically, it is desirable toprovide the recipient with a dosage of polyspecific immunoconjugate orantibody composite which is in the range of from about 1 pg/kg to 10mg/kg (amount of agent/body weight of patient), although a lower orhigher dosage also may be administered as circumstances dictate.

[0209] Administration of polyspecific immunoconjugates or antibodycomposites to a patient can be intravenous, intraarterial,intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal,by perfusion through a regional catheter, or by direct intralesionalinjection. When administering polyspecific immunoconjugates or antibodycomposites by injection, the administration may be by continuousinfusion or by single or multiple boluses.

[0210] Polyspecific immunoconjugates having a boron addend-loadedcarrier for thermal neutron activation therapy will normally be effectedin similar ways. However, it will be advantageous to wait untilnon-targeted polyspecific immunoconjugate clears before neutronirradiation is performed. Clearance can be accelerated using an antibodythat binds to the polyspecific immunoconjugate. See U.S. Pat. No.4,624,846 for a description of this general principle.

[0211] The polyspecific immunoconjugates and antibody composites of thepresent invention can be formulated according to known methods toprepare pharmaceutically useful compositions, whereby polyspecificimmunoconjugates or antibody composites are combined in a mixture with apharmaceutically acceptable carrier. A composition is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient patient. Sterile phosphate-buffered saline isone example of a pharmaceutically acceptable carrier. Other suitablecarriers are well-known to those in the art. See, for example,REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Ed. (1990).

[0212] For purposes of therapy, a polyspecific immunoconjugate (orantibody composite) and a pharmaceutically acceptable carrier areadministered to a patient in a therapeutically effective amount. Acombination of a polyspecific immunoconjugate (or antibody composite)and a pharmaceutically acceptable carrier is said to be administered ina “therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient. In the present context, an agent is physiologicallysignificant if its presence results in the inhibition of the growth oftarget cells, or in the increased susceptibility of target cells to achemotherapeutic agent.

[0213] Additional pharmaceutical methods may be employed to control theduration of action of a polyspecific immunoconjugate or antibodycomposite in a therapeutic application. Control release preparations canbe prepared through the use of polymers to complex or adsorb thepolyspecific immunoconjugate or antibody composite. For example,biocompatible polymers include matrices of poly(ethylene-co-vinylacetate) and matrices of a polyanhydride copolymer of a stearic aciddimer and sebacic acid. Sherwood et al., Bio/Technology 10: 1446 (1992).The rate of release of a polyspecific immunoconjugate (or antibodycomposite) from such a matrix depends upon the molecular weight of thepolyspecific immunoconjugate (or antibody composite), the amount ofpolyspecific immunoconjugate (or antibody composite) within the matrix,and the size of dispersed particles. Saltzman et al., Biophys. J. 55:163 (1989); Sherwood et al., supra. Other solid dosage forms aredescribed in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th ed. (1990).

[0214] The present invention also contemplates a method of treatment inwhich immunomodulators are administered to prevent, mitigate or reverseradiation-induced or drug-induced toxicity of normal cells, andespecially hematopoietic cells. Adjunct immunomodulator therapy allowsthe administration of higher doses of cytotoxic agents due to increasedtolerance of the recipient mammal. Moreover, adjunct immunomodulatortherapy can prevent, palliate, or reverse dose-limiting marrow toxicity.Examples of suitable immunomodulators for adjunct therapy include G-CSF,GM-CSF, thrombopoietin, IL-1, IL-3, and the like. The method of adjunctimmunomodulator therapy is disclosed by Goldenberg, U.S. Pat. No.5,120,525, which is incorporated by reference.

[0215] Those of skill in the art are aware that an antibody component isjust one example of a moiety that can be used to target particularcells. Other useful targeting moieties include non-antibody proteins,peptides, polypeptides, glycoproteins, lipoproteins, or the like, e.g.,growth factors, enzymes, receptor proteins, immunomodulators andhormones. For example, Sarkadi et al., The FASEB Journal 8: 766 (1994),which is incorporated by reference, provides methods for identifyinghydrophobic peptides that interact with P-glycoprotein. As anillustration, a polyspecific conjugate suitable for diagnosis and/ortreatment of certain multidrug resistant breast cancers would comprise ahydrophobic peptide that binds with P-glycoprotein, an epidermal growthfactor moiety that binds with the c-erb B2 proto-oncogene product, and adiagnostic or therapeutic agent.

[0216] The present invention, thus generally described, will beunderstood more readily by reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the present invention.

EXAMPLE 1 Production of Antibody Components: Murine P-Glycoprotein MAband Anti-CEA MAb

[0217] 1. Production of Monoclonal Antibodies

[0218] Methods for producing anti-P-glycoprotein murine monoclonalantibodies are well-known to those of skill in the art, as discussedabove. One approach is to immunize mice with cells, or cellularmembranes, that contain an abundance of P-glycoprotein. Cells thatover-express P-glycoprotein can be obtained by selecting and enrichingcells that express the MDR phenotype from a human continuous cell line.See, for example, Gottesman, “Drug-Resistant Mutants: Selection andDominance Analysis,” in METHODS IN ENZYMOLOGY, VOL. 151, Colowick etal., (eds.), pages 113-121 (Academic Press 1987), and Clynes et al.,Cytotechnology 12: 231 (1993).

[0219] A general method for using MDR cells to produceanti-P-glycoprotein monoclonal antibodies is described, for example, byRittmann-Grauer et al., Cancer Res. 52: 1810 (1992), which isincorporated by reference. Briefly, six week old female BALB/c mice areinjected intraperitoneally (i.p.) with 5×10⁶ MDR cells that have beenscraped from the surface of tissue culture flasks. Three weeks later,mice receive a second i.p. injection of 5×10⁶ MDR cells. Four days priorto fusion, mice receive a final intravenous boost of 5×10⁶ MDR cells.Splenocytes from the immunized mice are fused with murine myeloma cells,SP2/0-Ag 14, according to the method of Gerhard, “Fusion of Cells inSuspension and Outgrowth of Hybrids in Conditioned Medium,” inMONOCLONAL ANTIBODIES, Kennet et al. (eds.), pages 370-371 (PlenumPublishing Corp. 1981).

[0220] Anti-P-glycoprotein hybridoma cultures are initially screenedusing an indirect ELISA with a horseradish peroxidase conjugate of goatanti-mouse immunoglobulin. Monolayers of the MDR cells and thedrug-sensitive parental cell line are cultured in 96-well microtiterplates. Cells are fixed with 0.01% glutaraldehyde for 45 minutes at roomtemperature, the fixative is removed, cells are washed three times withphosphate-buffered saline (PBS), and the microtiter wells are blockedwith 10% bovine serum albumin for at least 45 minutes. Fifty microlitersof hybridoma supernatants are added to the microtiter wells and allowedto incubate for one hour at 37° C. Plates are then washed with PBS andincubated with 50 μl of peroxidase-conjugated goat anti-mouseimmunoglobulin diluted 1:1000 in PBS with 10% horse serum. Followingfive washes with PBS, positive clones are identified by the addition of100 μl of a solution containing 1 mg/ml O-phenylenediamine, 0.1%hydrogen peroxide, 50 mM citrate, and 100 mM sodium phosphate buffer (pH5.0). The reaction is quenched by the addition of 50 μl 4N sulfuricacid, and the plates are read at 490 nm.

[0221] Clones that produce a five-fold or greater ELISA signal for theMDR cells, compared with drug-sensitive cells, are expanded. Hybridomacells that produce anti-P-glycoprotein antibodies are injected intoBALB/c mice for ascites production according to the procedure ofHoogenraad et al., J. Immunol. Methods 61: 317 (1983).Anti-P-glycoprotein antibodies are purified from the ascites fluid usingprotein A chromatography. See, for example, Langone et al., J. Immunol.Methods 51: 3 (1982).

[0222] The production of highly specific anti-CEA MAb, has beendescribed by Hansen et al., Cancer 71: 3478 (1993), which isincorporated by reference. Briefly, a 20 gram BALB/c female mouse wasimmunized subcutaneously with 7.5 μg of partially-purified CEA incomplete Freund adjuvant. On day 3, the mouse was boosted subcutaneouslywith 7.5 μg of CEA in incomplete Freund adjuvant and then, the mouse wasboosted intravenously with 7.5 μg of CEA in saline on days 6 and 9. Onday 278, the mouse was given 65 μg of CEA intravenously in saline and 90μg of CEA in saline on day 404. On day 407, the mouse was sacrificed, acell suspension of the spleen was prepared, the spleen cells were fusedwith murine myeloma cells, SP2/0-Ag 14 (ATCC CRL 1581) usingpolyethylene glycol, and the cells were cultured in medium containing8-azaguanine. Hybridoma supernatants were screened for CEA-reactiveantibody using an ¹²⁵I-CEA radioimmunoassay (Roche; Nutley, N.J.).Positive clones were recloned.

[0223] 2. The Production of Antibody Fragments

[0224] As described above, proteolysis provides one method for preparingantibody fragments. This technique is well-known to those of skill inthe art. For example, see Coligan et al., supra, at pp. 2.8.1-2.8.10.Also see Stanworth et al. “Immunochemical Analysis of Human and RabbitImmunoglobulins and Their Subunits,” in HANDBOOK OF EXPERIMENTALIMMUNOLOGY, Vol. 1, Weir (ed.), pages 12.1-12.46 (Blackwell Scientific1986), and Parham, “Preparation and Purification of Active Fragmentsfrom Mouse Monoclonal Antibodies,” Id. at pages 14.1-14.23.

[0225] As an example, preactivated papain can be used to prepare F(ab)₂fragments from IgG1 or Fab fragments from IgG2a and IgG2b, as follows.Papain is activated by incubating 2 mg/ml papain (2×recrystallizedsuspension, Sigma #P3125) and 0.05 M cysteine (free-base, crystalline;Sigma #C7755) for 30 minutes in a 37° C. water bath. To remove cysteine,the papain/cysteine mixture is applied to a PD-10 column (Pharmacia#G-25), which has been equilibrated with 20 ml of acetate/EDTA buffer(0.1 M acetate with 3 mM EDTA, pH 5.5). Fractions are assayed bymeasuring absorbance at 280 nm, and the two or three fractions thatcontain protein are pooled. The concentration of preactivated papain isdetermined by using the formula: (absorbance at 280 nm)/2.5=mg.preactivated papain/ml.

[0226] To prepare antibody for digestion, 10 mg of antibody in 2 to 5 mlof PBS are dialyzed against acetate/EDTA buffer. Five hundred microgramsof preactivated papain are added to the dialyzed antibody solution, andthe mixture is vortexed. After a 6-12 hour incubation in a 37° C. waterbath, papain is inactivated by adding crystalline iodoacetamide (Sigma#I6125) to a final concentration of 0.03 M. The mixture is then dialyzedagainst 1 liter of PBS (pH 8.0) at 4° C. for 6-12 hours.

[0227] To remove undigested antibody and Fc fragments, the mixture isapplied to a protein A-Sepharose column which has been equilibrated inPBS (pH 8.0). Unbound fractions are collected in 2 ml aliquots andpooled. After concentrating the pool to a total volume of 5 ml or less,protein is fractionated by size-exclusion chromatography and the resultsare analyzed by SDS-PAGE.

EXAMPLE 2 Preparation of Antibody Composite:Anti-P-Glycoprotein/anti-CEA Bispecific Antibody

[0228] A bispecific F(ab′)₂ antibody composite is prepared from an Fab′fragment of an anti-P-glycoprotein monoclonal antibody and an Fab′fragment of a monoclonal antibody specific for CEA, using the methodsdescribed above. Also, see Goldenberg, international publication No. WO92/19273, which is incorporated by reference. Briefly, the interchaindisulfide bridges are reduced carefully with cysteine, taking care toavoid light-heavy chain cleavage, to form Fab′-SH fragments. The SHgroup(s) of one antibody fragment is(are) activated with an excess ofbis-maleimide linker (1,1′-(methylenedi-1,4-phenylene) bismaleimide(Aldrich Chemical Co.; Milwaukee, Wis.). The second antibody fragment isalso converted to Fab′-SH and then reacted with the activated firstantibody fragment to obtain a bispecific antibody composite.

EXAMPLE 3 Preparation of Polyspecific Immunoconjugate

[0229] A polyspecific immunoconjugate can be prepared by binding atherapeutic or diagnostic agent to the bispecific antibody composite,described in Example 2. As an example, the anti-P-glycoprotein/anti-CEAcomposite can be conjugated with doxorubicin via dextran, using themethod of Shih et al., Int. J. Cancer 41:832-839 (1988). Briefly, aminodextran is prepared by dissolving one gram of dextran (m.w. 18 kD; SigmaChemical Co.; St. Louis, Mo.) in 70 ml of water. The dextran ispartially oxidized to form polyaldehyde dextran by adding 0.5 gram ofsodium metaperiodate, and stirring the solution at room temperatureovernight. After concentrating the mixture with an Amicon cell (YM10membrane; MWCO=10,000), the polyaldehyde dextran is purified by SephadexG-25 chromatography and lyophilized to give about 900 grams of whitepowder. Polyaldehyde dextran is then treated with two equivalents of1,3-diamino-2-hydroxypropane in aqueous phase for 24 hours at roomtemperature. The resultant Schiff base is stabilized by addition ofsodium borohydride (0.311 mmol per 2.15 mmol of1,3-diamino-2-hydroxypropane) to the mixture. The mixture is allowed toincubate at room temperature for six hours. Amino dextran is purifiedusing a Sephadex G-25 column.

[0230] Doxorubicin (Sigma Chemical Co.; St. Louis, Mo.) is activated byadding one milliliter of anhydrous DMF to 0.1 mmole of doxorubicin in adried Reacti-vial, followed by a solution of N-hydroxysuccinimide (23mg, 0.2 mmole; Sigma) in 750 μl of anhydrous DMF and a solution of1,3-dicyclohexylcarbodiimide (41.5 mg, 0.2 mmol; Sigma) in 750 μl ofanhydrous DMF. The reaction mixture is stirred in the dark at roomtemperature for 16 hours under anhydrous conditions. The precipitate isthen centrifuged and the solution is stored in a sealed bottle at −20°C.

[0231] Doxorubicin-dextran intermediate conjugate is prepared bydissolving aminodextran (18 kD; 10 mg) in two milliliters of PBS (pH7.2) and gradually adding 0.7 ml of the aboveN-hydroxy-succinimide-activated doxorubicin solution. Thus, 50 moles ofdoxorubicin are present per mole of aminodextran. The solution isstirred at room temperature for five hours and after removing anyprecipitate, the conjugate is purified using a Sephadex G-25 column.Doxorubicin-dextran conjugate is typically characterized by adoxorubicin/dextran ratio of 14.

[0232] Alternatively, doxorubicin-dextran conjugate is prepared byreacting doxorubicin with 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide,as described by Shih et al., Int. J. Cancer 41:832-839 (1988). Also, seeShih et al., Cancer Research 51:4192-4198 (1991).

[0233] The bispecific antibody conjugate (25 mg) in 5 ml of PBS (pH 5.5)is oxidized in the dark by treatment with sodium metaperiodate (800 μlof a 21.5 mg/ml solution) at room temperature for 60 minutes. Thereaction mixture is then treated with ethylene glycol (50 μl) todecompose the unreacted periodate and the oxidized antibody fragment ispurified using a Sephadex G-25 column equilibrated in 0.05 M HEPES (pH7.4). Subsequently, the oxidized fragment is concentrated to 5 mg/ml in0.05 M HEPES (pH 7.4) and reacted with the doxorubicin-dextran conjugate(22 mg). After 24 hours at room temperature, the Schiff base is reducedby NaBH₃CN. Conjugated antibody is purified using a Sepharose CL-6Bcolumn.

EXAMPLE 4 Preparation of an Polyspecific Immunoconjugate Comprising aRadioisotope

[0234] A polyspecific immunoconjugate can be prepared in which aradioisotope is bound to one or more antibody components via a chelator.As an illustration, the antibody composite of Example 2 may beconjugated with either aminobenzyl diethylenetriaminepentaacetic acid(DTPA) or a derivative of DTPA containing the long-chain linker,—CSNH(CH₂)₁₀NH₂ (LC-DTPA). Briefly, the antibody composite (2.5 mg inabout one milliliter of 50 mM acetate-buffered 0.9% saline [ABS; pH5.3]) is oxidized in the dark by treatment with sodium metaperiodate(210 μl of a 5.68 mg/ml solution) at 0° C. for one hour. The reactionmixture is treated with ethylene glycol (20 μl) to decompose theunreacted periodate and the oxidized antibody fragment is purified usinga Sephadex G-50/80 column (Pharmacia; Piscataway, N.J.) equilibrated inPBS (pH 6.1). The oxidized fragment is then reacted with excess DTPA orLC-DTPA. After 40 hours at room temperature, the Schiff base is reducedby NaBH₃CN. Conjugated antibody composite is then purified using acentrifuged size-exclusion column (Sephadex G-50/80) equilibrated in 0.1M acetate (pH 6.5). The concentrations of antibody conjugates aredetermined by measuring absorbance at 280 nm.

[0235] The ratio of chelator molecules per molecule of antibodycomposite is determined by a metal-binding assay. The assay is performedby mixing an aliquot of the antibody conjugate with 0.1 M ammoniumacetate (pH 7) and 2 M triethanolamine, and incubating the mixture atroom temperature with a known excess of cobalt acetate spiked with⁵⁷cobalt acetate. After 30 minutes, EDTA (pH 7) is added to a finalconcentration of 10 mM. After a further 10 minute incubation, themixture is analyzed by instant thin layer chromatography (ITLC) using 10mM EDTA for development. The fraction of radioactivity bound to antibodyis determined by counting sections of ITLC strips on a gamma counter.Typically, the results will show that there are about 6 molecules ofDTPA per antibody component and about 5 molecules of LC-DTPA perantibody component.

[0236] Antibody conjugates are labeled with ⁹⁰yttrium, as follows.Briefly, commercially available ⁹⁰yttrium chloride (DuPont NEN; 17.68μl; 5.63 mCi) is buffered with 35.4 μl of 0.5 M acetate (pH 6.0). Thesolution is allowed to stand for 5-10 minutes at room temperature, andthen used for radiolabeling.

[0237]⁹⁰Yttrium-labeled antibody composite-DTPA is prepared by mixing⁹⁰yttrium acetate (128.7 μCi) with antibody composite-DTPA (30 μg; 8.3μl), incubating at room temperature for one hour, and diluting with 90μl of 0.1 M acetate (pH 6.5). ⁹⁰ Yttrium-labeled antibodycomposite-LC-DTPA is prepared by mixing ⁹⁰yttrium acetate (109.5 μCi)with antibody composite-LC-DTPA (30 μg; 7.6 μl), incubating at roomtemperature for one hour, and diluting with 90 μl of 0.1 M acetate (pH6.5).

[0238] The extent of ⁹⁰yttrium incorporation can be analyzed byincubating the labeling mixture with 10 mM EDTA for ten minutes,followed by ITLC examination using 10 mM EDTA for development. In thisassay, unbound ⁹⁰yttrium migrates with the solvent front, whileantibody-bound ⁹⁰yttrium remains at the origin. The presence of anycolloidal ⁹⁰yttrium is assayed by ITLC (co-spotted with human serumalbumin) using a water:ethanol:ammonia (5:2:1) solution for development.In this system, the fraction of radioactivity at the origin representscolloidal ⁹⁰yttrium. In addition, all labeling mixtures may be analyzedusing radio-high pressure liquid chromatography. Typically, 90 to 96% of⁹⁰yttrium is incorporated into the resultant polyspecificimmunoconjugate.

EXAMPLE 5 Treatment of Colon Cancer with ⁹⁰Yttrium-labeled PolyspecificImmunoconjugate and G-CSF

[0239] A patient has a carcinoembryonic antigen (CEA) blood titer of 55ng/ml due to peritoneal spread of a colon cancer which had been resectedtwo years earlier and found to be a Dukes' C lesion. Since previouschemotherapy with fluorouracil had been unsuccessful, the patientpresents for experimental therapy. The patient is given a 35 mCi dose ofthe ⁹⁰yttrium-labeled polyspecific immmunoconjugate prepared in Example4, by intraperitoneal injection. Two days later, an infusion of 5 μg/kgG-CSF (such as NEUPOGEN [Amgen, Inc.; Thousand Oaks, Calif.]) isinstituted intravenously, and the patient's hematologic values aremonitored thereafter. No significant drop in white blood cell count isnoted, thus permitting a repetition of the radioimmunotherapy threeweeks later, followed again by G-CSF therapy. A third treatment is giventwo months later, and radiological evidence of some tumor and ascitesreduction is noted four weeks later. Thus, the patient is able totolerate higher and more frequent doses of the radioimmunotherapy agent.

EXAMPLE 6 Preparation of an Antibody Composite Targeted to MultidrugResistant Psuedomonas Aeruginosa

[0240] Those of skill in the art can use standard methods to produceantibodies against a multidrug transporter protein of an infectiousagent. As an illustration, a bispecific antibody can be constructedwhich is targeted to multidrug resistant Psuedomonas aeruginosa.Antibody components that bind to OprK, a multidrug transporter proteinof Psuedomonas aeruginosa, can be obtained using OprK protein that isoverexpressed by bacterial cells. For example, the OprK gene can besynthesized using mutually priming long oligonucleotides which are basedupon the nucleotide sequence disclosed in Poole et al., J. Bacteriol.175: 7363 (1993). See, for example, Ausubel et al. (eds.), CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, pages 8.2.8 to 8.2.13 (WileyInterscience 1990). Also, see Wosnick et al., Gene 60:115 (1987).Moreover, current techniques using the polymerase chain reaction providethe ability to synthesize genes as large as 1.8 kilobases in length.Adang et al., Plant Molec. Biol. 21:1131 (1993); Bambot et al., PCRMethods and Applications 2:266 (1993).

[0241] The OprK gene is then cloned into a prokaryotic expression vectorwhich is subsequently introduced into competent E. coli cells, usingstandard techniques. See, for example, Ausubel et al., supra, at pages16.1.1-16.7.8. OprK protein is isolated from the host cells usingstandard techniques. (Id.)

[0242] Alternatively, OprK protein can be isolated from Psuedomonasaeruginosae which have been selected for the multidrug resistantphenotype, as described by Poole et al., supra.

[0243] Isolated OprK protein is used to generate anti-OprK MAb, asdescribed above. Also, see Mole et al., “Production of MonoclonalAntibodies Against Fusion Proteins Produced in Eschericia coli,” in DNACLONING, VOLUME III: A PRACTICAL APPROACH, Glover (ed.), pages 113-139(IRL Press 1987), and Dean “Preparation and Testing of MonoclonalAntibodies to Recombinant Proteins,₁” in METHODS IN MOLECULAR BIOLOGY,VOLUME 10: IMMUNOCHEMICAL PROTOCOLS, Manson (ed.) pages 43-63 (TheHumana Press, Inc. 1992).

[0244] Thus, anti-OprK MAb, or a fragment thereof, provides one antibodycomponent of a bispecific antibody. The second antibody component, whichbinds with a different antigen associated with the exterior surface ofPsuedomonas aeruginosa, may be obtained using the general techniquesdescribed above. Alternatively, suitable monoclonal antibodies can bepurchased from American Type Culture Collection (Rockville, Md.), suchas antibodies against Psuedomonas aeruginosa lipopolysaccharide (ATCCCRL Nos. 8753, 8754, 8795, 8796 and 8797), lipoprotein H2 of the outerenvelop of Psuedomonas aeruginosa (ATCC CRL 1783), Psuedomonasaeruginosa type a flagella (ATCC HB 9130), and Psuedomonas aeruginosatype b flagella (ATCC HB 9129).

[0245] An antibody composite comprising a moiety that binds OprK and amoiety that binds an exterior surface antigen of P. aeruginosa can beprepared using the methods described in Example 2.

EXAMPLE 7 Preparation and Use of an ¹¹¹lndium-labeled PolyspecificImmunoconjugate Targeted to Multidrug Resistant Psuedomonas Aeruginosa

[0246] Antibody composite-chelator conjugates are prepared as describedin Example 4. The conjugates are labeled with ¹¹¹Indium, as follows.Briefly, ¹¹¹Indium chloride is buffered at pH 5.5 using ammonium acetatesuch that the final acetate concentration is about 0.2 M. ¹¹¹Indiumacetate is added to a solution of the antibody composite-chelatorconjugate in 0.1 M acetate (pH 6.5), and the mixture is incubated forabout one hour. Typically, reaction mixtures contain either 10 μg ofantibody composite-DTPA and 73 μCi of ¹¹¹Indium, or 10 μg of antibodycomposite-LC-DTPA and 126.7 μCi of ¹¹¹Indium. The extent of ¹¹¹Indiumincorporation is analyzed using ITLC, as described above.

[0247] A patient with granulocytopenia has Pseudomonas aeruginosapneumonia which is no longer responsive to carbenicillin treatment. Fourmillicuries of ¹¹¹Indium-labeled polyspecific immmunoconjugate areinjected intravenously and after waiting at least 24 hours, the patientis scanned with a gamma camera. Foci of increased radioactivity appearas nodes in the lower lobes of the lung, indicating the presence ofpneumonic infiltrates with multidrug resistant Pseudomonas aeruginosa. Acourse of therapy is designed in which an aminoglycoside andcarbenicillin are administered with nonradioactive polyspecificimmunoconjugate that comprises an OprK-binding moiety, a moiety thatbinds an exterior surface antigen of Pseudomonas aeruginosa and achemosensitizing agent.

EXAMPLE 8 Preparation of a ^(99m)Tc-labeled Polyspecific ImmunoconjugateTargeted to Multidrug Resistant Psuedomonas Aeruginosa

[0248] An antibody composite is prepared which binds oprK and E87antigen, an exterior surface antigen of Psuedomonas aeruginosa. Generaltechniques for preparing the antibody composite are described in Example6, and preparation of anti-E87 monoclonal antibodies is described bySawada et al., U.S. Pat. No. 5,089,262.

[0249] The antibody composite is labeled with ^(99m)Tc using methodsthat are well-known to those of skill in the art. See, for example,Crockford et al., U.S. Pat. No. 4,424,200, Paik et al., U.S. Pat. No.4,652,440, Baidoo et al., Cancer Research (Suppl.) 50: 799s (1990),Griffiths et al., Cancer Research 51: 4594 (1991), Pak et al., U.S. Pat.No. 5,053,493, Griffiths et al., U.S. Pat. No. 5,128,119, Lever et al.,U.S. Pat. No. 5,095,111, and Dean et al., U.S. Pat. No. 5,180,816.

[0250] As an illustration, ^(99m)Tc-labeled polyspecific immunoconjugatecan be obtained as described by Hansen et al., U.S. Pat. No. 5,328,679.Briefly, a solution of 0.075 M SnCl₂ (solution I) is prepared bydissolving 3350 mg SnCl.2H₂O in one milliliter of 6 N HCl and dilutingthe resultant solution with sterile H₂O which has been purged withargon. A solution of 0.1 M NaK tartrate in 0.05 M NaAc (pH 5.5)[solution II] is prepared with sterile H₂O purged with argon. One volumeof solution I is mixed with 26 volumes of solution II, and the resultantsolution III is filter sterilized and purged with argon.

[0251] A solution of antibody composite is reduced with 20 mM cysteine,and excess cysteine is removed by gel filtration. The reduced antibodycomposite (2 mg/ml) is stabilized at pH 4.5 in 0.05 M NaOAc buffercontaining 0.15 M saline. The resultant solution IV is filter sterilizedand purged with argon. Solution IV is mixed with a sufficient amount ofsolution III to obtain a final concentration of 123 μg Sn per mg ofreduced antibody composite. The resultant solution V is adjusted to a pHof 4.5-4.8.

[0252] A sterile solution of sodium pertechnetate (10 mCi) in saline isadded to an aliquot of solution V which contains 1.25 mg antibodycomposite and stable stannous ions, and the mixture is gently agitated.Labeling is quantitative within 5 minutes. The resultant solution of^(99m)Tc-labeled polyspecific immunoconjugate is ready for immediateinjection.

[0253] The ^(99m)Tc-labeled polyspecific immunoconjugate is administeredto a subject, and sites of infection caused by multidrug resistantPsuedomonas aeruginosa are localized using single-photon emissioncomputed tomography.

[0254] Although the foregoing refers to particular preferredembodiments, it will be understood that the present invention is not solimited. It will occur to those of ordinary skill in the art thatvarious modifications may be made to the disclosed embodiments and thatsuch modifications are intended to be within the scope of the presentinvention, which is defined by the following claims.

[0255] All publications and patent applications mentioned in thisspecification are indicative of the level of skill of those in the artto which the invention pertains. All publications and patentapplications are herein incorporated by reference to the same extent asif each individual publication or patent application were specificallyand individually indicated to be incorporated by reference in itsentirety.

What is claimed is:
 1. A polyspecific immunoconjugate comprising: (a) atleast one antibody component that binds with a first epitope of amultidrug transporter protein; (b) at least one antibody component thatbinds with a first epitope of an antigen, wherein said antigen isassociated with a tumor or an infectious agent; and (c) at least onediagnostic or therapeutic agent.
 2. The polyspecific immunoconjugate ofclaim 1, wherein said antibody components are selected from the groupconsisting of: (a) a murine monoclonal antibody; (b) a humanizedantibody derived from (a); (c) a human monoclonal antibody; (d) asubhuman primate antibody; and (e) an antibody fragment derived from(a), (b), (c) or (d).
 3. The polyspecific immunoconjugate of claim 2,wherein said antibody fragment is selected from the group consisting ofF(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv and minimal recognition unit.
 4. Thepolyspecific immunoconjugate of claim 3, wherein said multidrugtransporter protein is selected from the group consisting ofP-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr,EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC,Bmr, TetA and OprK.
 5. The polyspecific immunoconjugate of claim 4,wherein said diagnostic agent is selected from the group consisting ofradioactive label, photoactive agent or dye, florescent label, enzymelabel, bioluminescent label, chemiluminescent label, colloidal gold andparamagnetic ion.
 6. The polyspecific immunoconjugate of claim 5,wherein said radioactive label is selected from the group consisting ofγ-emitters and positron-emitters.
 7. The polyspecific immunoconjugate ofclaim 6, wherein said γ-emitters have a gamma radiation emission peak inthe range of 50-500 Kev.
 8. The polyspecific immunoconjugate of claim 7,wherein said γ-emitters with a gamma radiation emission peak in therange of 50-500 Kev are selected from the group consisting of ^(99m)Tc,⁶⁷Ga, ¹²³I, ¹²⁵I and ¹³¹I.
 9. The polyspecific immunoconjugate of claim4, wherein said therapeutic agent is selected from the group consistingof radioisotope, boron addend, immunomodulator, toxin, photoactive agentor dye, cancer chemotherapeutic drug, antiviral drug, antifungal drug,antibacterial drug, antiprotozoal drug and chemosensitizing agent. 10.The polyspecific immunoconjugate of claim 9, wherein said radioisotopeis selected from the group consisting of α-emitters, β-emitters,γ-emitters, Auger electron emitters, neutron capturing agents that emita particles and radioisotopes that decay by electron capture.
 11. Thepolyspecific immunoconjugate of claim 9, wherein said radioisotope isselected from the group consisting of ¹⁹⁸Au, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y,¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu and ²¹¹At.
 12. The polyspecific immunoconjugate ofclaim 4, further comprising an antibody component that binds with asecond epitope of said multidrug transporter protein.
 13. Thepolyspecific immunoconjugate of claim 12, further comprising an antibodycomponent that binds with a second epitope of said tumor or infectiousagent associated antigen, or with an epitope of a second antigenassociated with said tumor or said infectious agent.
 14. Thepolyspecific immunoconjugate of any one of claims 4, 12 or 13, furthercomprising an immunomodulator, wherein said immunomodulator is selectedfrom the group consisting of cytokines, stem cell growth factors andhematopoietic factors.
 15. A method for treating a mammal having eithera multidrug resistant tumor that expresses a tumor associated antigen ora multidrug resistant disease caused by an infectious agent, said methodcomprising the step of administering a polyspecific immunoconjugate tothe mammal, wherein said polyspecific immunoconjugate comprises: (a) atleast one antibody component that binds with a first epitope of amultidrug transporter protein, (b) at least one antibody component thatbinds with a first epitope of an antigen, wherein said antigen isassociated with said tumor or said infectious agent, and (c) at leastone therapeutic agent.
 16. The method of claim 15, wherein said antibodycomponents are selected from the group consisting of: (a) a murinemonoclonal antibody; (b) a humanized antibody derived from (a); (c) ahuman monoclonal antibody; (d) a subhuman primate antibody; and (e) anantibody fragment derived from (a), (b), (c) or (d).
 17. The method ofclaim 16, wherein said antibody fragment is selected from the groupconsisting of F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv and minimalrecognition unit.
 18. The method of claim 15, wherein said multidrugtransporter protein is selected from the group consisting ofP-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr,EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC,Bmr, TetA and OprK.
 19. The method of claim 18, wherein said therapeuticagent is selected from the group consisting of radioisotope, boronaddend, toxin, immunomodulator, photoactive agent or dye, cancerchemotherapeutic drug, antiviral drug, antifungal drug, antibacterialdrug, antiprotozoal drug and a chemosensitizing agent.
 20. The method ofclaim 19, wherein said radioisotope is selected from the groupconsisting of α-emitters, β-emitters, γ-emitters, Auger electronemitters, neutron capturing agents that emit α-particles andradioisotopes that decay by electron capture.
 21. The method of claim19, wherein said radioisotope is selected from the group consisting of¹⁹⁸Au, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu and ²¹¹At.
 22. Themethod of claim 19, further comprising the step of administering achemosensitizing agent to said mammal.
 23. The method of claim 19,wherein said therapeutic agent is a chemosensitizing agent.
 24. Themethod of claim 23, further comprising the step of administering achemotherapeutic agent selected from the group consisting of cancerchemotherapeutic drug, antibacterial drug, antiviral drug, antifungaldrug and antiprotozoal drug.
 25. The method of claim 19, furthercomprising the step of administering an immunomodulator, wherein saidimmunomodulator is selected from the group consisting of cytokine, stemcell growth factor and hematopoietic factor.
 26. The method of claim 25,wherein said cytokine is granulocyte-colony stimulating factor.
 27. Themethod of claim 25, wherein said hematopoietic factor is thrombopoietin.28. The method of claim 25, wherein said immunomodulator is administeredprior to or simultaneously with said administration of said polyspecificimmunoconjugate.
 29. The method of claim 25, wherein saidimmunomodulator is administered subsequent to said administration ofsaid polyspecific immunoconjugate.
 30. A method for detecting thelocation of multidrug resistant (MDR) tumor cells, MDR HIV-infectedcells or MDR infectious agents in a mammal having a multidrug resistantdisease caused by a tumor or infectious agent, said method comprisingthe steps of: (a) parenterally injecting the mammal with an antibodycomposite comprising (1) at least one antibody component that binds afirst epitope of a multidrug transporter protein, and (2) at least oneantibody component that binds a first epitope of an antigen that isassociated with the tumor or the infectious agent, wherein said antibodycomposite is conjugated with a biotin-binding-molecule or with biotin;(b) parenterally injecting a clearing composition comprised of: (i)biotin, when said antibody composite is conjugated with a biotin-bindingmolecule, or (ii) a biotin-binding molecule, when said antibodycomposite is conjugated with biotin, and allowing said clearingcomposition to substantially clear said antibody composite from sitesthat do not contain MDR tumor cells, MDR HIV-infected cells or MDRinfectious agents; and (c) parenterally injecting a diagnosticcomposition comprised of: (i) biotin, when said antibody composite isconjugated with a biotin-binding molecule, or (ii) a biotin-bindingmolecule, when said antibody composite is conjugated with biotin, and adiagnostic agent which is conjugated with said biotin or saidbiotin-binding molecule.
 31. The method of claim 30, wherein saidantibody components are selected from the group consisting of: (a) amurine monoclonal antibody; (b) a humanized antibody derived from (a);(c) a human monoclonal antibody; (d) a subhuman primate antibody; and(e) an antibody fragment derived from (a), (b), (c) or (d).
 32. Themethod of claim 31, wherein said antibody fragment is selected from thegroup consisting of F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv and minimalrecognition unit.
 33. The method of claim 32, wherein said multidrug Xtransporter protein is selected from the group consisting ofP-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr,EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC,Bmr, TetA and OprK.
 34. The method of claim 33, wherein said diagnosticagent is selected from the group consisting of radioactive label,photoactive agent or dye, fluorescent label and paramagnetic ion. 35.The method of claim 34, wherein said radioactive label is selected fromthe group consisting of γ-emitters and positron-emitters.
 36. The methodof claim 35, wherein said γ-emitters have a gamma radiation emissionpeak in the range of 50-500 Kev.
 37. The method of claim 36, whereinsaid γ-emitters with a gamma radiation emission peak in the range of50-500 Kev are selected from the group consisting of ^(99m)Tc, ⁶⁷Ga,¹²³I, ¹²⁵I and ¹³¹I.
 38. The method of claim 37, wherein saidbiotin-binding molecule is avidin or streptavidin.
 39. A method fortreating a mammal having a multidrug resistant disease caused by a tumoror infectious agent, said method comprising the steps of: (a)parenterally injecting the mammal with an antibody composite comprising(1) at least one antibody component that binds a first epitope of amultidrug transporter protein, and (2) at least one antibody componentthat binds a first epitope of an antigen that is associated with thetumor or the infectious agent, wherein said antibody composite isconjugated with a biotin-binding molecule or with biotin; (b)parenterally injecting a clearing composition comprised of: (i) biotin,when said antibody composite is conjugated with a biotin-bindingmolecule, or (ii) a biotin-binding molecule, when said antibodycomposite is conjugated with biotin, and allowing said clearingcomposition to substantially clear said antibody composite from sitesthat do not contain multidrug resistant (MDR) cells or MDR infectiousagents; and (c) parenterally injecting, a therapeutic compositioncomprised of: (i) biotin, when said antibody composite is conjugatedwith a biotin-binding molecule, or (ii) a biotin-binding molecule, whensaid antibody composite is conjugated with biotin, and a therapeuticagent which is conjugated with said biotin or said biotin-bindingmolecule.
 40. The method of claim 39, wherein said biotin-bindingmolecule is avidin or streptavidin.
 41. The method of claim 40, whereinsaid antibody components are selected from the group consisting of: (a)a murine monoclonal antibody; (b) a humanized antibody derived from (a);(c) a human monoclonal antibody; (d) a subhuman primate antibody; and(e) an antibody fragment derived from (a), (b), (c) or (d). 42.The-method of claim 41, wherein said antibody fragment is selected fromthe group consisting of F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv and minimalrecognition unit.
 43. The method of claim 42, wherein said multidrugtransporter protein is selected from the group consisting ofP-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr,EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC,Bmr, TetA and OprK.
 44. The method of claim 43, wherein said therapeuticagent is selected from the group consisting of radioisotope, boronaddend, toxin, immunomodulator, photoactive agent or dye, cancerchemotherapeutic drug, antiviral drug, antifungal drug, antibacterialdrug, antiprotozoal drug and a chemosensitizing agent.
 45. The method ofclaim 44, wherein said radioisotope is selected from the groupconsisting of α-emitters, β-emitters, γ-emitters, Auger electronemitters, neutron capturing agents that emit α-particles andradioisotopes that decay by electron capture.
 46. The method of claim44, wherein said radioisotope is selected from the group consisting of¹⁹⁸Au, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu and ²¹¹At.
 47. A methodfor detecting the presence of multidrug resistant (MDR) tumor cells, MDRHIV-infected cells or MDR infectious agents in a mammal, said methodcomprising: (a) removing from the mammal a biological sample that issuspected of containing MDR tumor cells, MDR HIV-infected cells or MDRinfectious agents; (b) contacting said biological sample with anantibody composite which comprises (1) at least one antibody componentthat binds with a first epitope of a multidrug transporter protein, and(2) at least one antibody component that binds with a first epitope ofan antigen that is associated with said tumor or said infectious agent,wherein said contacting is performed under conditions which allow thebinding of said antibody composite to said biological sample; and (c)detecting any of said bound antibody composite.
 48. The method of claim47, wherein said antibody components are selected from the groupconsisting of: (a) a murine monoclonal antibody; (b) a humanizedantibody derived from (a); (c) a human monoclonal antibody; (d) asubhuman primate antibody; and (e) an antibody fragment derived from(a), (b), (c) or (d).
 49. The method of claim 48, wherein said antibodyfragment is selected from the group consisting of F(ab′)₂, F(ab)₂, Fab′,Fab, Fv, sFv and minimal recognition unit.
 50. The method of claim 49,wherein said multidrug transporter protein is selected from the groupconsisting of P-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA,QacA, CmlA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA,DrrA, DrrB, TlrC, Bmr, TetA and OprK.
 51. The method of claim 50,wherein said antibody composite further comprises a diagnostic agentselected from the group consisting of radioisotope, fluorescent label,chemiluminescent label, enzyme label, bioluminescent label and colloidalgold.
 52. The method of claim 50, wherein said antibody compositefurther comprises biotin or a biotin-binding molecule.
 53. A method fordetecting the location of multidrug resistant (MDR) tumor cells, MDRHIV-infected cells or MDR infectious agents in a mammal having amultidrug resistant disease caused by a tumor or infectious agent, saidmethod comprising the steps of: (a) parenterally injecting the mammalwith a polyspecific immunoconjugate that comprises (1) at least oneantibody component that binds with a first epitope of a multidrugtransporter protein, (2) at least one antibody component that binds witha first epitope of an antigen that is associated with the tumor orinfectious agent, and (3) a diagnostic agent; (b) parenterally injectingsaid mammal with an antibody or antibody fragment that binds with saidpolyspecific immunoconjugate in an amount that is sufficient to decreasethe level of circulating polyspecific immunoconjugate by about 10-85%within 2 to 72 hours; (c) scanning said mammal with a detector to locatethe site or sites of uptake of said polyspecific immunoconjugate. 54.The method of claim 53, wherein said antibody components are selectedfrom the group consisting of: (a) a murine monoclonal antibody; (b) ahumanized antibody derived from (a); (c) a human monoclonal antibody;(d) a subhuman primate antibody; and (e) an antibody fragment derivedfrom (a), (b), (c) or (d).
 55. The method of claim 54, wherein saidantibody fragment is selected from the group consisting of F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, sFv and minimal recognition unit.
 56. The methodof claim 55, wherein said multidrug transporter protein is selected fromthe group consisting of P-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA,NorA, QacA, CmlA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC,MsrA, DrrA, DrrB, TlrC, Bmr, TetA and OprK.
 57. The method of claim 56,wherein said diagnostic agent is selected from the group consisting ofradioactive label, photoactive agent or dye, fluorescent label andparamagnetic ion.
 58. The method of claim 57, wherein said radioactivelabel is selected from the group consisting of γ-emitters andpositron-emitters.
 59. The method of claim 58, wherein said γ-emittershave a gamma radiation emission peak in the range of 50-500 Kev.
 60. Themethod of claim 59, wherein said γ-emitters with a gamma radiationemission peak in the range of 50-500 Kev are selected from the groupconsisting of ^(99m)Tc, ⁶⁷Ga, ¹²³I, ¹²⁵I and ¹³¹I.
 61. A method fortreating a mammal having a multidrug resistant disease caused by a tumoror infectious agent, said method comprising the steps of: (a)parenterally injecting the mammal with a polyspecific immunoconjugatecomprising (1) at least one antibody component that binds with a firstepitope of a multidrug transporter protein, (2) at least one antibodycomponent that binds with a first epitope of an antigen that isassociated with the tumor or infectious agent, and (3) a therapeuticagent; and (b) parenterally injecting said mammal with an antibody orantibody fragment that binds with said polyspecific immunoconjugate inan amount that is sufficient to decrease the level of circulatingpolyspecific immunoconjugate by about 10-85% within 2 to 72 hours. 62.The method of claim 61, wherein said antibody components are selectedfrom the group consisting of: (a) a murine monoclonal antibody; (b) ahumanized antibody derived from (a); (c) a human monoclonal antibody;(d) a subhuman primate antibody; and (e) an antibody fragment derivedfrom (a), (b), (c) or (d).
 63. The method of claim 62, wherein saidantibody fragment is selected from the group consisting of F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, sFv and minimal recognition unit.
 64. The methodof claim 63, wherein said multidrug transporter protein is selected fromthe group consisting of P-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA,NorA, QacA, CmlA, Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC,MsrA, DrrA, DrrB, TlrC, Bmr, TetA and OprK.
 65. The method of claim 64,wherein said therapeutic agent is selected from the group consisting ofradioisotope, boron addend, toxin, immunomodulator, photoactive agent ordye, cancer chemotherapeutic drug, antiviral drug, antifungal drug,antibacterial drug, antiprotozoal drug and a chemosensitizing agent. 66.The method of claim 65, wherein said radioisotope is selected from thegroup consisting of α-emitters, β-emitters, γ-emitters, Auger electronemitters, neutron capturing agents that emit α-particles andradioisotopes that decay by electron capture.
 67. The method of claim65, wherein said radioisotope is selected from the group consisting of¹⁹⁸Au, ³²P, ¹²⁵I, ¹³¹I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu and ²¹¹At.
 68. A methodfor detecting the location of multidrug resistant (MDR) tumor cells, MDRHIV-infected cells or MDR infectious agents in a subject having amultidrug resistant disease caused by a tumor or infectious agent, saidmethod comprising the steps of: (a) parenterally injecting the subjectwith a polyspecific immunoconjugate comprising (1) at least one antibodycomponent that binds with a first epitope of a multidrug transporterprotein, (2) at least one antibody component that binds with a firstepitope of an antigen that is associated with a tumor or infectiousagent, and (3) a diagnostic agent; (b) surgically exposing orendoscopically accessing the interior of the body cavity of saidsubject; and (c) scanning said interior body cavity with a detectionprobe to detect the sites of accretion of said polyspecificimmunoconjugate.
 69. The method of claim 68, wherein said antibodycomponents are selected from the group consisting of: (a) a murinemonoclonal antibody; (b) a humanized antibody derived from (a); (c) ahuman monoclonal antibody; (d) a subhuman primate antibody; and (e) anantibody fragment derived from (a), (b), (c) or (d).
 70. The method ofclaim 69, wherein said antibody fragment is selected from the groupconsisting of F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv and minimalrecognition unit.
 71. The method of claim 70, wherein said multidrugtransporter protein is selected from the group consisting ofP-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr,EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC,Bmr, TetA and OprK.
 72. The method of claim 71, wherein said diagnosticagent is a radioisotope.
 73. The method of claim 72, wherein saidradioisotope is a γ-emitter or a positron-emitter.
 74. The method ofclaim 72, wherein said diagnostic agent is a photoactive agent or dye.75. The method of claim 74, wherein said photoactive agent or dye isdetected by laser-induced fluorescence.
 76. An antibody compositecomprising: (a) at least one antibody component that binds with a firstepitope of a multidrug transporter protein; and (b) at least oneantibody component that binds with a first epitope of an antigen,wherein said antigen is associated with a tumor or an infectious agent.77. The antibody composite of claim 76, wherein said antibody componentsare selected from the, group consisting of: (a) a murine monoclonalantibody; (b) a humanized antibody derived from (a); (c) a humanmonoclonal antibody; (d) a subhuman primate antibody; and (e) anantibody fragment derived from (a), (b), (c) or (d).
 78. The antibodycomposite of claim 77, wherein said antibody fragment is selected fromthe group consisting of F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv and minimalrecognition unit.
 79. The antibody composite of claim 78, wherein saidmultidrug transporter protein is selected from the group consisting ofP-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr,EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC,Bmr, TetA and OprK.
 80. The antibody composite of claim 79, furthercomprising an antibody component that binds with a second epitope ofsaid multidrug transporter protein.
 81. The antibody composite of claim80, further comprising an antibody component that binds with a secondepitope of said tumor or infectious agent associated antigen, or with anepitope of a second antigen associated with said tumor or saidinfectious agent.
 82. A method for treating a mammal having either amultidrug resistant tumor that expresses a tumor associated antigen or amultidrug resistant disease caused by an infectious agent, said methodcomprising the step of administering an antibody composite to themammal, wherein said antibody composite comprises: (a) at least oneantibody component that binds with a first epitope of a multidrugtransporter protein, and (b) at least one antibody component that bindswith a first epitope of an antigen, wherein said antigen is associatedwith said tumor or said infectious agent.
 83. The method of claim 82,wherein said antibody components are selected from the group consistingof: (a) a murine monoclonal antibody; (b) a humanized antibody derivedfrom (a); (c) a human monoclonal antibody; (d) a subhuman primateantibody; and (e) an antibody fragment derived from (a), (b), (c) or(d).
 84. The method of claim 83, wherein said antibody fragment isselected from the group consisting of F(ab′)₂, F(ab)₂, Fab′, Fab, Fv,sFv and minimal recognition unit.
 85. The method of claim 84, whereinsaid multidrug transporter protein is selected from the group consistingof P-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA,Bcr, EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB,TlrC, Bmr, TetA and OprK.
 86. The method of claim 85, further comprisingthe step of administering a therapeutic agent to said mammal, whereinsaid therapeutic agent is selected from the group consisting of cancerchemotherapeutic drug, antiviral drug, antifungal drug, antibacterialdrug and antiprotozoal drug.
 87. The method of claim 86, furthercomprising the step of administering an immunomodulator, wherein saidimmunomodulator is selected from the group consisting of cytokine, stemcell growth factor and hematopoietic factor.
 88. The method of claim 87,wherein said cytokine is granulocyte-colony stimulating factor.
 89. Themethod of claim 87, wherein said hematopoietic factor is thrombopoietin.90. The method of claim 87, wherein said immunomodulator is administeredprior to or simultaneously with said administration of said antibodycomposite.
 91. The method of claim 87, wherein said immunomodulator isadministered subsequent to said administration of said antibodycomposite.
 92. A method for treating a subject having a multidrugresistant disease caused by a tumor or infectious agent, said methodcomprising the steps of: (a) parenterally injecting the subject with apolyspecific immunoconjugate comprising (1) at least one antibodycomponent that binds with a first epitope of a multidrug transporterprotein, (2) at least one antibody component that binds with a firstepitope of an antigen that is associated with a tumor or infectiousagent, and (3) a photoactive agent or dye; (b) surgically exposing orendoscopically accessing the interior of the body cavity of saidsubject;, and (c) treating sites of accretion of said polyspecificimmunoconjugate to light, wherein said treatment activates saidphotoactive agent or dye.
 93. The method of claim 92, wherein saidantibody components are selected from the group consisting of: (a) amurine monoclonal antibody; (b) a humanized antibody derived from (a);(c) a human monoclonal antibody; (d) a subhuman primate antibody; and(e) an antibody fragment derived from (a), (b), (c) or (d).
 94. Themethod of claim 93, wherein said antibody fragment is selected from thegroup consisting of F(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv and minimalrecognition unit.
 95. The method of claim 94, wherein said multidrugtransporter protein is selected from the group consisting ofP-glycoprotein, OtrB, Tel(L), Mmr, ActII, TcmA, NorA, QacA, CmlA, Bcr,EmrB, EmrD, AcrE, EnvD, MexB, Smr, QacE, MvrC, MsrA, DrrA, DrrB, TlrC,Bmr, TetA and OprK.